IL1-Beta Binding Antibodies and Fragments thereof

ABSTRACT

An IL-1β binding antibody or IL-1β binding fragment thereof comprising the amino acid sequence of SEQ ID NO: 8 and SEQ ID NO: 9, and related nucleic acids, vectors, cells, and compositions, as well as method of using same to treat or prevent a disease. IL-1β binding antibodies or IL-1β binding fragments thereof are provided which have desirable affinity and potency.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 11/472,813filed Jun. 21, 2006, which claims priority of U.S. provisionalapplication Ser. No. 60/692,830 filed Jun. 21, 2005, the disclosures ofwhich are incorporated herein by reference.

FIELD OF INVENTION

The invention relates to IL-1β binding antibodies and includingfragments thereof, and nucleic acids encoding such antibodies, as wellas to vectors, cells, and compositions comprising the antibodies ornucleic acids, and uses thereof.

BACKGROUND OF THE INVENTION

The interleukin-1 (IL-1) family of cytokines has been implicated indisease states such as rheumatoid arthritis (RA), osteoarthritis,Crohn's disease, ulcerative colitis (UC), septic shock, chronicobstructive pulmonary disease (COPD), asthma, graft versus host disease,atherosclerosis, adult T-cell leukemia, multiple myeloma, multiplesclerosis, stroke, and Alzheimer's disease. IL-1 family members includeIL-1α, IL-1β, and IL-1Ra. Although related by their ability to bind toIL-1 receptors (IL-1R1 and IL-1R2), each of these cytokines is expressedby a different gene and has a different primary amino acid sequence.Furthermore, the physiological activities of these cytokines can bedistinguished.

Compounds that disrupt IL-1 receptor signaling have been investigated astherapeutic agents to treat IL-1 mediated diseases. These compoundsinclude recombinant IL-1Ra (Amgen Inc., Thousand Oaks, Calif.) and IL-1receptor “trap” peptide (Regeneron Inc., Tarrytown, N.Y.).Animal-derived monoclonal antibodies that bind IL-1 cytokines also havebeen investigated. However, their clinical value can be limited due totheir immunogenicity. For example, human subjects administered withmouse monoclonal antibodies have been known to produce human anti-mouseantibodies (HAMA). HAMA have been reported to reduce the efficacy ofmonoclonal antibody therapy and to produce adverse reactions, includingkidney damage. Other IL-1β antibodies may be limited by their bindingaffinity and/or their potency. Accordingly, additional compounds thatdisrupt IL-1 receptor signaling are needed. The invention provides suchcompounds, as well as methods for preparing and using such compounds.

BRIEF SUMMARY

The invention provides an IL-1β binding antibody or IL-1β bindingfragment thereof comprising the amino acid sequence of SEQ ID NO: 2.Also provided herein is a nucleic acid encoding the antibody or antibodyfragment, as well as a vector comprising the nucleic acid, a cellcomprising the nucleic acid or vector, and a composition comprising theantibody, nucleic acid, or vector.

The invention further provides a method of treating or preventing adisease in a mammal comprising administering an effective amount of anantibody or antibody fragment, nucleic acid, or vector of the inventionto a mammal in need thereof, whereby a disease is treated or preventedin the mammal.

The invention provides a method of preparing an affinity matured IL-1βbinding polypeptide comprising (a) providing a first nucleic acidcomprising a nucleic acid sequence encoding an IL-1β binding polypeptidethat comprises the amino acid sequence of any of SEQ ID NOS: 1-26 and asecond nucleic acid comprising a nucleic acid sequence that differs fromthe first nucleic acid sequence by at least one nucleotide, (b)performing nucleic acid shuffling to provide two or more mutated nucleicacids, (c) selecting for a mutated nucleic acid that encodes apolypeptide that (i) binds to IL-1β with a greater affinity than thepolypeptide encoded by the first nucleic acid, (ii) has a selectivityfor IL-1β over IL-1α that is greater than that of the polypeptideencoded by the first nucleic acid, (iii) has an equilibrium bindingdissociation constant (K_(D)) for IL-1β that is lower than that of thepolypeptide encoded by the first nucleic acid, or (iv) inhibits IL-1βinduced expression of serum IL-6 in an animal to a greater degree thanthe polypeptide encoded by the first nucleic acid, and (d) expressingthe selected mutated nucleic acid, whereby an affinity matured IL-1βbinding polypeptide is produced.

The invention provides novel IL-1β binding antibodies or IL-1β bindingfragments thereof, which bind to human IL-1β with a dissociationconstant lower than 3 pM, alternatively about 2 pM or less, preferablyabout 1 pM or less. Such high affinity antibodies are contemplated asbeing useful for various methods of treating or preventing IL-1 relateddiseases or conditions. Alternatively or additionally, the IL-1β bindingantibodies or IL-1β binding fragments bind to an IL-1β epitope such thatthe bound antibody or fragment does not substantially prevent the IL-1βfrom binding to IL-1 receptor type I. Alternatively or additionally, theIL-1β binding antibodies or IL-1β binding fragments bind tosubstantially the same epitope as one or more of the exemplaryantibodies described herein, such as the antibody designated AB7 whichcomprises a heavy chain variable region. Alternatively or additionally,the IL-1β binding antibodies or IL-1β binding fragments compete with thebinding of an antibody having the light chain variable region of SEQ IDNO:11 and the heavy chain variable region of SEQ ID NO:15. Alternativelyor additionally, the present invention encompasses IL-1β bindingantibodies or IL-1β binding fragments that bind to an epitope containedin the sequence ESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO:36). Exemplary IL-1βbinding antibodies include the antibodies designated AB5 and AB7 herein.

The invention also provides IL-1 binding antibodies or IL-1β bindingfragments thereof having a dissociation constant of less than 3 pM,alternatively about 1 pM or less, alternatively any of the otherdissociation constants disclosed herein, and comprising a heavy chainvariable region comprising one of the amino acid sequences of SEQ ID NO:12, 13, 21, 23 or 24, or alternatively the amino acid sequence of SEQ IDNO: 12, 13 or 21, or alternatively the amino acid sequence of SEQ ID NO:13 or 21, or alternatively the amino acid sequence of SEQ ID NO: 8, 14or 15, or alternatively the amino acid sequence of SEQ ID NO: 8 or 15.The IL-1β binding antibody or IL-1β binding fragment can also compriselight chain variable region comprising the amino acid sequence of SEQ IDNO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

As another embodiment of the present invention, novel IL-1β bindingantibodies, or IL-1β binding fragments thereof, are provided which bindIL-1β with a dissociation constant between about 6 pM and about 50 pM,alternatively between about 13 pM and about 25 pM, alternatively about19 pM, and where the antibody or fragment has an IC₅₀ less than 0.5 nM(500 pM), alternatively between about 5 pM and about 200 pM,alternatively between about 10 pM and about 100 pM, alternatively about30 pM, for inhibiting IL-1β stimulated release of IL-6 from humanfibroblasts. IC₅₀ for inhibiting IL-1β stimulated release of IL-6 fromhuman fibroblasts refers the concentration required to inhibit 50% ofIL-6 released by IL-1β stimulation of the human fibroblasts. Exemplaryantibodies include the antibody designated AB1 herein.

The present invention also provides IL-1β binding antibodies or IL-1βbinding fragments thereof having a dissociation constant between about 6and about 50 pM and comprising a heavy chain variable region comprisingone of the amino acid sequences of SEQ ID NOS: 4, 5 or 6, alternativelyone of the amino acid sequences of SEQ ID NOS: 4 or 5, alternatively theamino acid sequences of SEQ ID NO: 4. It is contemplated that in somecircumstances, an IL-1β binding antibody or IL-1β binding fragmenthaving a relatively higher dissociation constant may be desirable, forexample, for some methods of treating or preventing IL-1 relateddiseases or conditions where a relatively lower degree of affinity isdesirable.

Exemplary antibodies include the antibodies designated AB1, AB2, AB3,AB4, AB5, AB6, AB7, AB8, and AB9. AB1 comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:4 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:9.AB2 comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:5 and a light chain variable region comprising theamino acid sequence of SEQ ID NO:9. AB3 comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:6 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:9.AB4 comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:7 and a light chain variable region comprising theamino acid sequence of SEQ ID NO:9. AB5 comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:8 and a lightchain variable region comprising the amino acid sequence of SEQ ID NO:9.AB6 comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:14 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:10. AB7 comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:15 and alight chain variable region comprising the amino acid sequence of SEQ IDNO:11. AB8 comprises has a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:25 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:10. AB9 comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:26 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:11.

The present invention encompasses IL-1β binding antibodies or IL-1βbinding fragments having a heavy chain variable region that comprisesany one of the sequences set forth in SEQ ID NO:2, 4-8, 12-15, 21,23-26, 28-35, or 42-57, alternatively any one of the sequences set forthin SEQ ID NO: 21, alternatively any one of the sequences set forth inSEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8,alternatively any one of the sequences set forth in SEQ ID NO: 12 or SEQID NO: 13, alternatively any one of the sequences set forth in SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 25, or SEQ ID NO: 26.

The invention also encompasses IL-1β binding antibodies or IL-1β bindingfragments having a light chain variable region that comprises any one ofthe sequences set forth in SEQ ID NO: 1, 9-11, or 27, alternatively anyone of the sequences set forth SEQ ID NO: 1, alternatively any one ofthe sequences set forth in SEQ ID NO: 9, alternatively any one of thesequences set forth in SEQ ID NO: 10 or SEQ ID NO: 11.

The present invention also encompasses IL-1β antibodies or IL-1β bindingfragments comprising one of the heavy chain variable regions of thesequences set forth in SEQ ID NO:2, 4-8, 12-15, 21, 23-26, 28-35, or42-57, and one of the light chain variable regions of the sequences setforth in SEQ ID NO:1, 9-11, or 27.

The present invention also encompasses IL-1β binding antibodies or IL-1βbinding fragments comprising portions that do not bind IL-1β but insteadare responsible for other functions, such as circulating half-life,direct cytotoxic effect, detectable labeling, or activation of arecipient's endogenous complement cascade or endogenous cellularcytotoxicity. Antibodies of the invention may comprise all or a portionof a constant region of an antibody. The constant region may be selectedfrom any isotype, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. For example, the antibody maycomprise an IgG2 region. In addition to, or instead of, comprising aconstant region, the antibodies and fragments of the invention mayinclude an epitope tag, a salvage receptor epitope, a label moiety fordiagnostic or purification purposes, or a cytotoxic moiety such as aradionuclide or toxin.

The present invention also encompasses pharmaceutical compositionscomprising any one of the IL-1β binding antibodies or IL-1β bindingfragments and a pharmaceutically suitable carrier, excipient or diluent.Preferably the antibodies and compounds of the invention can beadministered in a therapeutically effective amount, that is, an amountsufficient to ameliorate a clinical sign or symptom of a condition ordisorder associated with the target protein expression, to a subject inneed of such treatment. In a related embodiment, the pharmaceuticalcomposition further comprises a second active agent. In yet anotherrelated embodiment, the pharmaceutical composition is provided whereinthe second active agent is an antibody to or antagonist of growth factoror, a cytokine. In another embodiment the second active agent is anotherantibody.

In another embodiment of the present invention, the use of the IL-1βantibodies or IL-1β binding fragments is contemplated in the manufactureof a medicament for preventing or reducing a condition or disorderassociated with IL-1. In any of the uses, the medicament can becoordinated with treatment using a second active agent. In anotherembodiment of the invention, the use of a synergistic combination of anantibody of the invention for preparation of a medicament for treating apatient exhibiting symptoms of a IL-1β related condition or disorderdisclosed herein wherein the medicament is coordinated with treatmentusing a second active agent is contemplated. In a related embodiment,the second active agent is an antibody to or antagonist of cytokine or,a growth factor. Embodiments of any of the aforementioned uses arecontemplated wherein the amount of the IL-1β binding antibody orfragment in the medicament is at a dose effective to reduce the dosageof second active agent required to achieve a therapeutic effect.

Kits are also contemplated by the present invention. In one embodiment,a kit comprises a therapeutically or prophylactically effective amountof a compound or composition of the invention (such as an antibody,fragment, nucleic acid, vector or cell), packaged in a container, suchas a vial or bottle, and further comprising a label attached to orpackaged with the container, the label describing the contents of thecontainer and providing indications and/or instructions regarding use ofthe contents of the container to prevent or reduce a condition ordisease associated with target protein expression.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a pair of amino acid sequences corresponding to the lightchains and heavy chains of the variable region of some of the antibodiesdescribed herein. The underlined portions of the amino acid sequencesindicate complementarity determining regions (CDRs).

FIG. 2 is a set of amino acid sequences corresponding to the light chainand heavy chain variable regions of antibodies AB1, AB2, AB3, and AB4.The underlined portions of the amino acid sequences indicatecomplementarity determining regions (CDRs).

FIG. 3 is a set of amino acid sequences corresponding to the light chainand heavy chain variable regions of antibodies AB5, AB5.1, and AB5.2.The underlined portions of the amino acid sequences indicatecomplementarity determining regions (CDRs).

FIG. 4 is a set of amino acid sequences corresponding to the light chainand heavy chain variable regions of antibodies AB5.3 and AB5.4. Theunderlined portions of the amino acid sequences indicate complementaritydetermining regions (CDRs).

FIG. 4A is a set of amino acid sequences corresponding to the lightchain and heavy chain variable regions of antibodies AB6 and AB7. Theunderlined portions of the amino acid sequences indicate complementaritydetermining regions (CDRs).

FIG. 4B is a set of amino acid sequences corresponding to the lightchain and heavy chain variable regions of antibodies AB8 and AB9. Theunderlined portions of the amino acid sequences indicate complementaritydetermining regions (CDRs).

FIG. 5 is graph showing the results of an in vitro IL-1β stimulationexperiment.

FIG. 6 is a histogram showing the results of an in vivo IL-1βstimulation experiment.

FIG. 7 is a graph showing kinetic exclusion assay results for theantibody designated AB1.

FIG. 8 is a graph showing kinetic exclusion assay results for theantibody designated AB5.

FIG. 9 is a graph showing kinetic exclusion assay results for theantibody designated AB7.

FIG. 10 is a graph showing the results of an in vitro IL-1β stimulationexperiment for the antibodies designated AB1, AB2, and AB3.

FIG. 11 is a graph showing the results of an in vitro IL-1β stimulationexperiment for the antibodies designated AB1 and AB7.

FIG. 12 is a graph showing the results of an in vitro IL-1β stimulationexperiment for the antibodies designated AB5 and AB7, as well as forKineret®.

FIG. 13 is a histogram showing the results of an in vivo IL-1βstimulation experiment for the antibodies designated AB5 and AB1.

FIG. 14 is a histogram showing the results of an in vivo stimulationexperiment for the antibodies designated AB5 and AB7.

FIG. 15 is a Western blot showing the results of cross-reactivityexperiments for the antibody designated AB7 with IL-1β from cynomolgusmonkey and rhesus macaque.

FIG. 16 is a Western blot showing the results of cross-reactivityexperiments for the antibody designated AB7 with IL-1β from dog, guinea,pig, and rabbit.

FIG. 17 is a Western blot showing the results of cross-reactivityexperiments for the antibody designated AB7 with recombinant human,mouse, and rat IL-1β.

FIG. 18 is a graph showing the results of an in vitro experiment for theantibody designated AB7 and for Kineret involving IL-1 inducedproduction of IL-8.

FIG. 19 is a graph showing the results of an assay to examine whetherthe present antibodies prevent IL-1β from binding to IL-1 receptor typeI.

FIG. 20 is an illustration of an assay to examine whether the presentantibodies prevent IL-1β from binding to IL-1 receptor type I.

DETAILED DESCRIPTION

The present invention encompasses novel IL-1β antibodies and fragmentshaving desirable affinity and potency. As one aspect of the presentinvention, IL-1β binding antibodies are provided which have unexpectedlyhigh affinity and low dissociation constants (for example, less than 3pM, alternatively about 1 pM or less) compared to known IL-1β bindingantibodies. Exemplary antibodies include the antibodies designated AB5and AB7 herein. As another aspect of the present invention, IL-1βbinding antibodies are provided having a desirable dissociation constant(for example, between about 6 pM and about 50 pM) and a desirable IC₅₀(for example, less than 500 pM) for inhibiting IL-1β stimulated releaseof IL-6 from human fibroblasts. Exemplary antibodies include theantibody designated AB1 herein.

The present invention also encompasses IL-1β binding antibodies or IL-1βbinding fragments that bind selectively to IL-1β in that they bind toIL-1β with greater affinity than to other antigens. The IL-1β bindingantibodies or IL-1β binding fragments may bind selectively to humanIL-1β, but also bind detectably to non-human IL-1. Alternatively oradditionally, the antibodies or fragments may bind to human IL-1β and toIL-1β of at least one other mammal (a first mammal) and not to IL-1β ofat least one other mammal (a second mammal). For example, the antibodiesor fragments may bind to one or more of rodent IL-1β, primate IL-1β, dogIL-1β, and rabbit IL-1β, and/or not bind to guinea pig IL-1β.Alternatively or additionally, the antibodies or fragments may bind tomouse IL-1β with higher affinity than to rat IL-1β. Alternatively oradditionally, the IL-1β binding antibodies or IL-1β binding fragmentsmay have the same or substantially the same potency against human IL-1βand primate IL-1β. Alternatively or additionally, the IL-1β bindingantibodies or IL-1β binding fragments may have the same or substantiallythe same potency against recombinant human IL-1β and endogenous humanIL-1β. Alternatively or additionally, the IL-1β binding antibodies orIL-1β binding fragments may neutralize mouse IL-1β.

As used herein, an antibody or fragment that specifically binds with atarget antigen refers to an antibody that binds the target antigen withgreater affinity than with similar antigens. For example, an antibody orfragment is specific for its cognate antigen when the variable regionsof the antibody or fragment recognize and bind the cognate antigen witha detectable preference (distinguishing the antigen from other knownpolypeptides of the same family, by virtue of measurable differences inbinding affinity, despite the possible existence of localized sequenceidentity, homology, or similarity between family members). It will beunderstood that specific antibodies and fragments may also interact withother proteins (for example, S. aureus protein A or other antibodies inELISA techniques) through interactions with sequences outside thevariable region of the antibodies, and in particular, in the constantregion of the antibody or fragment. Screening assays to determinebinding specificity of an antibody are well known and routinelypracticed in the art. For a comprehensive discussion of such assays, seeHarlow et al. (Eds), Antibodies A Laboratory Manual; Cold Spring HarborLaboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6.

An aspect of the present invention encompasses IL1β binding antibodiesand IL1β binding fragments thereof having unexpectedly low dissociationconstants (K_(D)), for example, less than 3 pM, alternatively 2 pM orless, alternatively 1 pM or less, alternatively 0.8 pM or less,alternatively 0.74 pM or less, alternatively 0.72 pM or less,alternatively 0.7 pM or less, alternatively 0.6 pM or less,alternatively 0.56 pM or less, alternatively 0.5 pM or less,alternatively 0.3 pM or less, alternatively 0.26 pM or less,alternatively 0.24 pM or less, alternatively 0.2 pM or less. Thus, insome embodiments of the present invention, IL1β binding antibodies andfragments may be described by reference to a high end of a range ofdissociation constants. Additionally or alternatively, in someembodiments of the present invention, IL1β binding antibodies andfragments may be described by reference to a low end of a range ofdissociation constants, such as for example, an antibody or fragmenthaving a dissociation constant of 0.07 pM or higher, alternatively 0.1pM or higher, alternatively 0.11 pM or higher, alternatively 0.15 pM orhigher, alternatively 0.2 pM or higher, alternatively 0.24 pM or higher,alternatively 0.26 pM or higher, alternatively 0.3 pM or higher,alternatively 0.5 pM or higher, alternatively 0.7 pM or higher. Anyhigher dissociation constant and lower dissociation constant, asspecified above, may be combined to define a range of dissociationconstants, providing that the lower value selected is equal to or lessthan the higher value selected.

Another aspect of the present invention provides novel IL-1β bindingantibodies, and IL-1β binding fragments thereof, which bind IL-1β with adissociation constant greater than 6 pM and less than or equal to 50 pM,alternatively between about 13 pM and about 25 pM, and where theantibody or fragment has an IC₅₀ for inhibiting IL-1β stimulated releaseof IL-6 from human fibroblasts that is less than 0.5 nM (500 pM),alternatively between about 5 pM and about 200 pM, alternatively betweenabout 10 pM and about 100 pM, alternatively about 30 pM. It iscontemplated that it may be desirable to provide an IL-1β bindingantibody or fragment having the foregoing binding affinity and potencyfor some methods of treating or preventing IL-1β mediated conditions ordiseases. Exemplary antibodies include the antibody designated AB1herein.

The present antibodies and fragments bind to IL-1β with high affinity,as indicated by the dissociation constants set forth herein. Affinityconstants characterizing the affinities of antibodies to antigens may beassociation constants measured by the kinetics of antigen-antibodycomplex formation. Alternatively, binding affinity may be characterizedby a dissociation constant which is the inverse of the associationconstant. The term K_(D), as used herein, is intended to refer to thedissociation constant of an antibody-antigen interaction.

The present invention also encompasses neutralizing antibodies orneutralizing fragments thereof which bind to IL-1β so as to neutralizebiological activity of the IL-1. Neutralization of biological activityof IL-1β can be assessed by assays for one or more indicators of IL-1βbiological activity, such as IL-1β stimulated release of IL-6 from humanfibroblasts or other cells, IL-1β induced release of IL-8 from bloodcells, or IL-1 induced proliferation of T helper cells. Preferably theIL-1β binding antibodies and fragments of the present inventionneutralize the biological activity of IL-1β connected with the signalingfunction of IL-1 receptor type I (IL-1RI) bound by the IL-1β. Ingeneral, the neutralizing antibodies and fragments of the presentinvention can neutralize the biological activity of IL-1, regardless ofwhether the binding of IL-1β to IL1 receptor type I is blocked. Morepreferably, the IL-1β binding antibodies or IL-1β binding fragmentsneutralize the biological activity of IL-1β by binding to IL-1, withoutsubstantially preventing the binding of the bound IL-1β to IL-1 receptortype I. A potential advantage of such antibodies and fragments is thatthey can bind and neutralize IL-1β while still permitting the IL-1β tobind to IL-1RE. This can result in an effective reduction in IL-1αbiological activity as well as IL-1β biological activity, since thereare fewer unbound IL-1RI sites for IL-1α to bind to. Thus, IL-1β bindingantibodies and fragments of the present invention are useful in methodswhere it is desirable to neutralize IL-1 biological activity in vitro anin vivo.

The present antibodies or fragments may be neutralizing antibodies orfragments which bind specifically to IL-1β epitope that affectsbiological activity of IL-1.

The present antibodies or fragments can bind to aneutralization-sensitive epitope of IL-1. When aneutralization-sensitive epitope of IL-1β is bound by one of the presentantibodies or fragments, the result is a loss of biological activity ofthe IL-1β containing the epitope.

In some embodiments, the IL-1β binding antibodies or IL-1β bindingfragments may have an IC₅₀ for inhibiting IL-1β stimulated release ofIL-1β from blood cells that is less than 50 pM, alternatively about 25pM or less, alternatively about 10 pM or less, alternatively about 2 pMor less. IC₅₀ for inhibiting IL-1β stimulated release of IL-1β fromblood cells refers the concentration required to inhibit 50% of IL-8released by IL-1β stimulation of blood cells. Exemplary antibodiesinclude the antibody designated AB7 herein.

The present invention also encompasses an IL-1β binding antibody orIL-1β binding fragment thereof, comprising a changed amino acidsequence, wherein the changed amino acid has one or at least onesubstitution, addition or deletion from a starting amino acid sequenceselected from SEQ ID NOS:27 or 28 (or any of the other sequencesdisclosed herein), where the changed antibody or fragment has the sameor substantially the same affinity and specificity of epitope binding asthe starting amino acid sequence. It is contemplated that one or moresubstitutions, deletions, or additions may be made to the IL-1β bindingantibodies or IL-1β binding fragments provided herein, such asantibodies or fragments comprising SEQ ID NO:28 and/or SEQ ID NO:27,while maintaining the same or substantially the same affinity andspecificity of epitope binding of the starting antibody or fragment. Forexample, the present invention encompasses an IL-1β binding antibody orIL-1β binding fragment thereof, comprising a changed amino acidsequence, wherein the changed amino acid has one or at least onesubstitution, addition or deletion from a starting amino acid sequencecomprising SEQ ID NO:8 (or any of the other sequences disclosed hereincan be used as a starting sequence), where the changed antibody orfragment has the same or substantially the same affinity and specificityof epitope binding as the starting amino acid sequence comprising SEQ IDNO:8 (or the particular sequence that is used as the starting amino acidsequence). By the phrase “substantially the same” affinity, it is meantthat the affinity or dissociation constant as determined by theteachings herein, is not increased or decreased more than inherentvariation in the assay for an antibody or fragment comprising SEQ IDNOS:28 or 27, such as the variation observed when the assay is performedthree or more independent times. By the phrase “substantially the same”epitope specificity, it is meant that binding to an amino acid sequencecontaining the epitope as determined by the teachings herein is withininherent variation in the assay for an antibody or fragment comprisingSEQ ID NOS:28 or 27, such as the variation observed when performed threeor more independent times. When comparing to an antibody or fragmentcomprising SEQ ID NOS:28 or 27, it is meant that the comparison shouldbe made between the changed amino acid sequence and the starting aminoacid sequence from which the one or more substitutions, deletions, oradditions were made, such starting sequence being identical at all otheramino acids.

Antibodies, Humanized Antibodies, and Human Engineered Antibodies

The IL-1β binding antibodies of the present invention may be provided aspolyclonal antibodies, monoclonal antibodies (mAbs), recombinantantibodies, chimeric antibodies, CDR-grafted antibodies, fully humanantibodies, single chain antibodies, and/or bispecific antibodies, aswell as fragments, including variants and derivatives thereof, providedby known techniques, including, but not limited to enzymatic cleavage,peptide synthesis or recombinant techniques.

Antibodies generally comprise two heavy chain polypeptides and two lightchain polypeptides, though single domain antibodies having one heavychain and one light chain and heavy chain antibodies devoid of lightchains are also contemplated. There are five types of heavy chains,called alpha, delta, epsilon, gamma and mu, based on the amino acidsequence of the heavy chain constant domain. These different types ofheavy chains give rise to five classes of antibodies, IgA (includingIgA₁ and IgA₂), IgD, IgE, IgG and IgM, respectively, including foursubclasses of IgG, namely IgG₁, IgG₂, IgG₃ and IgG₄. There are also twotypes of light chains, called kappa (κ) or lambda (λ) based on the aminoacid sequence of the constant domains. A full-length antibody includes aconstant domain and a variable domain. The constant region need not bepresent in an antigen binding fragment of an antibody. Antigen bindingfragments of an antibody disclosed herein can include Fab, Fab′,F(ab′)₂, and F(v) antibody fragments. As discussed in more detail below,IL-1β binding fragments encompass antibody fragments and antigen-bindingpolypeptides that will bind IL-1β.

Each of the heavy chain and light chain sequences of an antibody, orantigen binding fragment thereof, includes a variable region with threecomplementarity determining regions (CDRs) as well as non-CDR frameworkregions (FRs). The terms “heavy chain” and “light chain,” as usedherein, mean the heavy chain variable region and the light chainvariable region, respectively, unless otherwise noted. Heavy chain CDRsare referred to herein as CDR-H1, CDR-H2, and CDR-H3. Light chain CDRsare referred to herein as CDR-L1, CDR-L2, and CDR-L3. Variable regionsand CDRs in an antibody sequence can be identified (i) according togeneral rules that have been developed in the art or (ii) by aligningthe sequences against a database of known variable regions. Methods foridentifying these regions are described in Kontermann and Dubel, eds.,Antibody Engineering, Springer, New York, N.Y., 2001, and Dinarello etal., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken,N.J., 2000. Databases of antibody sequences are described in and can beaccessed through “The Kabatman” database at www.bioinf.org.uk/abs(maintained by A. C. Martin in the Department of Biochemistry &Molecular Biology University College London, London, England) and VBASE2at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33(Database issue): D671-D674 (2005). The “Kabatman” database web sitealso includes general rules of thumb for identifying CDRs. The term“CDR,” as used herein, is as defined in Kabat et al., Sequences ofImmunological Interest, 5^(th) ed., U.S. Department of Health and HumanServices, 1991, unless otherwise indicated.

The present invention encompasses IL-1β binding antibodies that includetwo full length heavy chains and two full length light chains.Alternatively, the IL-1β binding antibodies can be constructs such assingle chain antibodies or “mini” antibodies that retain bindingactivity to IL-1β. Such constructs can be prepared by methods known inthe art such as, for example, the PCR mediated cloning and assembly ofsingle chain antibodies for expression in E. coli (as described inAntibody Engineering, The practical approach series, J. McCafferty, H.R. Hoogenboom, and D. J. Chiswell, editors, Oxford University Press,1996). In this type of construct, the variable portions of the heavy andlight chains of an antibody molecule are PCR amplified from cDNA. Theresulting amplicons are then assembled, for example, in a second PCRstep, through a linker DNA that encodes a flexible protein linkercomposed of the amino acids Gly and Ser. This linker allows the variableheavy and light chain portions to fold in such a way that the antigenbinding pocket is regenerated and antigen is bound with affinities oftencomparable to the parent full-length dimeric immunoglobulin molecule.

The IL-1β binding antibodies and fragments of the present inventionencompass variants of the exemplary antibodies, fragments and sequencesdisclosed herein. Variants include peptides and polypeptides comprisingone or more amino acid sequence substitutions, deletions, and/oradditions that have the same or substantially the same affinity andspecificity of epitope binding as one or more of the exemplaryantibodies, fragments and sequences disclosed herein. Thus, variantsinclude peptides and polypeptides comprising one or more amino acidsequence substitutions, deletions, and/or additions to the exemplaryantibodies, fragments and sequences disclosed herein where suchsubstitutions, deletions and/or additions do not cause substantialchanges in affinity and specificity of epitope binding. For example, avariant of an antibody or fragment may result from one or more changesto an antibody or fragment comprising one or more of amino acid sequenceof SEQ ID NOS:1-35 or 42-57, where the changed antibody or fragment hasthe same or substantially the same affinity and specificity of epitopebinding as the starting sequence. Variants may be naturally occurring,such as allelic or splice variants, or may be artificially constructed.Variants may be prepared from the corresponding nucleic acid moleculesencoding said variants. Variants of the present antibodies and IL-1βbinding fragments may have changes in light and/or heavy chain aminoacid sequences that are naturally occurring or are introduced by invitro engineering of native sequences using recombinant DNA techniques.Naturally occurring variants include “somatic” variants which aregenerated in vivo in the corresponding germ line nucleotide sequencesduring the generation of an antibody response to a foreign antigen.

Variants of IL-1β binding antibodies and IL-1β binding fragments mayalso be prepared by mutagenesis techniques. For example, amino acidchanges may be introduced at random throughout an antibody coding regionand the resulting variants may be screened for binding affinity forIL-1β or for another property. Alternatively, amino acid changes may beintroduced in selected regions of an IL-1β antibody, such as in thelight and/or heavy chain CDRs, and/or in the framework regions, and theresulting antibodies may be screened for binding to IL-1β or some otheractivity. Amino acid changes encompass one or more amino acidsubstitutions in a CDR, ranging from a single amino acid difference tothe introduction of multiple permutations of amino acids within a givenCDR, such as CDR3. In another method, the contribution of each residuewithin a CDR to IL-1β binding may be assessed by substituting at leastone residue within the CDR with alanine. Lewis et al. (1995), Mol.Immunol. 32: 1065-72. Residues which are not optimal for binding toIL-1β may then be changed in order to determine a more optimum sequence.Also encompassed are variants generated by insertion of amino acids toincrease the size of a CDR, such as CDR3. For example, most light chainCDR3 sequences are nine amino acids in length. Light chain sequences inan antibody which are shorter than nine residues may be optimized forbinding to IL-1β by insertion of appropriate amino acids to increase thelength of the CDR.

Variants may also be prepared by “chain shuffling” of light or heavychains. Marks et al. (1992), Biotechnology 10: 779-83. A single light(or heavy) chain can be combined with a library having a repertoire ofheavy (or light) chains and the resulting population is screened for adesired activity, such as binding to IL-1β. This permits screening of agreater sample of different heavy (or light) chains in combination witha single light (or heavy) chain than is possible with librariescomprising repertoires of both heavy and light chains.

The IL-1β binding antibodies and fragments of the present inventionencompass derivatives of the exemplary antibodies, fragments andsequences disclosed herein. Derivatives include polypeptides orpeptides, or variants, fragments or derivatives thereof, which have beenchemically modified. Examples include covalent attachment of one or morepolymers, such as water soluble polymers, N-linked, or O-linkedcarbohydrates, sugars, phosphates, and/or other such molecules. Thederivatives are modified in a manner that is different from naturallyoccurring or starting peptide or polypeptides, either in the type orlocation of the molecules attached. Derivatives further include deletionof one or more chemical groups which are naturally present on thepeptide or polypeptide.

The IL-1β binding antibodies and fragments of the present invention canbe bispecific. Bispecific antibodies or fragments can be of severalconfigurations. For example, bispecific antibodies may resemble singleantibodies (or antibody fragments) but have two different antigenbinding sites (variable regions). Bispecific antibodies can be producedby chemical techniques (Kranz et al. (1981), Proc. Natl. Acad. Sci. USA,78: 5807), by “polydoma” techniques (U.S. Pat. No. 4,474,893) or byrecombinant DNA techniques. Bispecific antibodies of the presentinvention can have binding specificities for at least two differentepitopes, at least one of which is an epitope of IL-1β. The IL-1βbinding antibodies and fragments can also be heteroantibodies.Heteroantibodies are two or more antibodies, or antibody bindingfragments (Fab) linked together, each antibody or fragment having adifferent specificity.

Techniques for creating recombinant DNA versions of the antigen-bindingregions of antibody molecules which bypass the generation of monoclonalantibodies are contemplated for the present IL-1β binding antibodies andfragments. DNA is cloned into a bacterial expression system. One exampleof such a technique suitable for the practice of this invention uses abacteriophage lambda vector system having a leader sequence that causesthe expressed Fab protein to migrate to the periplasmic space (betweenthe bacterial cell membrane and the cell wall) or to be secreted. Onecan rapidly generate and screen great numbers of functional Fabfragments for those which bind IL-1β. Such IL-1β binding agents (Fabfragments with specificity for an IL-1β polypeptide) are specificallyencompassed within the IL-1β binding antibodies and fragments of thepresent invention.

The present IL-1β binding antibodies and fragments can be humanized orhuman engineered antibodies. As used herein, a humanized antibody, orantigen binding fragment thereof, is a recombinant polypeptide thatcomprises a portion of an antigen binding site from a non-human antibodyand a portion of the framework and/or constant regions of a humanantibody. A human engineered antibody or antibody fragment is anon-human (e.g., mouse) antibody that has been engineered by modifying(e.g., deleting, inserting, or substituting) amino acids at specificpositions so as to reduce or eliminate any detectable immunogenicity ofthe modified antibody in a human.

Humanized antibodies include chimeric antibodies and CDR-graftedantibodies. Chimeric antibodies are antibodies that include a non-humanantibody variable region linked to a human constant region. Thus, inchimeric antibodies, the variable region is mostly non-human, and theconstant region is human. Chimeric antibodies and methods for makingthem are described in Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6841-6855 (1984), Boulianne, et al., Nature, 312: 643-646 (1984), andPCT Application Publication WO 86/01533. Although, they can be lessimmunogenic than a mouse monoclonal antibody, administrations ofchimeric antibodies have been associated with human immune responses(HAMA) to the non-human portion of the antibodies. Chimeric antibodiescan also be produced by splicing the genes from a mouse antibodymolecule of appropriate antigen-binding specificity together with genesfrom a human antibody molecule of appropriate biological activity, suchas the ability to activate human complement and mediate ADCC. Morrisonet al. (1984), Proc. Natl. Acad. Sci., 81: 6851; Neuberger et al.(1984), Nature, 312: 604. One example is the replacement of a Fc regionwith that of a different isotype.

CDR-grafted antibodies are antibodies that include the CDRs from anon-human “donor” antibody linked to the framework region from a human“recipient” antibody. Generally, CDR-grafted antibodies include morehuman antibody sequences than chimeric antibodies because they includeboth constant region sequences and variable region (framework) sequencesfrom human antibodies. Thus, for example, a CDR-grafted humanizedantibody of the invention can comprise a heavy chain that comprises acontiguous amino acid sequence (e.g., about 5 or more, 10 or more, oreven 15 or more contiguous amino acid residues) from the frameworkregion of a human antibody (e.g., FR-1, FR-2, or FR-3 of a humanantibody) or, optionally, most or all of the entire framework region ofa human antibody. CDR-grafted antibodies and methods for making them aredescribed in, Jones et al., Nature, 321: 522-525 (1986), Riechmann etal., Nature, 332: 323-327 (1988), and Verhoeyen et al., Science, 239:1534-1536 (1988)). Methods that can be used to produce humanizedantibodies also are described in U.S. Pat. Nos. 4,816,567, 5,721,367,5,837,243, and 6,180,377. CDR-grafted antibodies are considered lesslikely than chimeric antibodies to induce an immune reaction againstnon-human antibody portions. However, it has been reported thatframework sequences from the donor antibodies are required for thebinding affinity and/or specificity of the donor antibody, presumablybecause these framework sequences affect the folding of theantigen-binding portion of the donor antibody. Therefore, when donor,non-human CDR sequences are grafted onto unaltered human frameworksequences, the resulting CDR-grafted antibody can exhibit, in somecases, loss of binding avidity relative to the original non-human donorantibody. See, e.g., Riechmann et al., Nature, 332: 323-327 (1988), andVerhoeyen et al., Science, 239: 1534-1536 (1988).

Human engineered antibodies include “veneered” antibodies and antibodiesprepared using HUMAN ENGINEERING™ technology (XOMA (US) LLC, Berkeley,Calif.). HUMAN ENGINEERING™ technology is commercially available, andinvolves altering an non-human antibody or antibody fragment, such as amouse or chimeric antibody or antibody fragment, by making specificchanges to the amino acid sequence of the antibody so as to produce amodified antibody with reduced immunogenicity in a human thatnonetheless retains the desirable binding properties of the originalnon-human antibodies. Generally, the technique involves classifyingamino acid residues of a non-human (e.g., mouse) antibody as “low risk”,“moderate risk”, or “high risk” residues. The classification isperformed using a global risk/reward calculation that evaluates thepredicted benefits of making particular substitution (e.g., forimmunogenicity in humans) against the risk that the substitution willaffect the resulting antibody's folding and/or antigen-bindingproperties. Thus, a low risk position is one for which a substitution ispredicted to be beneficial because it is predicted to reduceimmunogenicity without significantly affecting antigen bindingproperties. A moderate risk position is one for which a substitution ispredicted to reduce immunogenicity, but is more likely to affect proteinfolding and/or antigen binding. High risk positions contain residuesmost likely to be involved in proper folding or antigen binding.Generally, low risk positions in a non-human antibody are substitutedwith human residues, high risk positions are rarely substituted, andhumanizing substitutions at moderate risk positions are sometimes made,although not indiscriminately. Positions with prolines in the non-humanantibody variable region sequence are usually classified as at leastmoderate risk positions.

The particular human amino acid residue to be substituted at a given lowor moderate risk position of a non-human (e.g., mouse) antibody sequencecan be selected by aligning an amino acid sequence from the non-humanantibody's variable regions with the corresponding region of a specificor consensus human antibody sequence. The amino acid residues at low ormoderate risk positions in the non-human sequence can be substituted forthe corresponding residues in the human antibody sequence according tothe alignment. Techniques for making human engineered proteins aredescribed in greater detail in Studnicka et al., Protein Engineering, 7:805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and5,869,619, and PCT Application Publication WO 93/11794.

“Veneered” antibodies are non-human or humanized (e.g., chimeric orCDR-grafted antibodies) antibodies that have been engineered to replacecertain solvent-exposed amino acid residues so as to further reducetheir immunogenicity or enhance their function. As surface residues of achimeric antibody are presumed to be less likely to affect properantibody folding and more likely to elicit an immune reaction, veneeringof a chimeric antibody can include, for instance, identifyingsolvent-exposed residues in the non-human framework region of a chimericantibody and replacing at least one of them with the correspondingsurface residues from a human framework region. Veneering can beaccomplished by any suitable engineering technique, including the use ofthe above-described HUMAN ENGINEERING™ technology.

In a different approach, a recovery of binding avidity can be achievedby “de-humanizing” a CDR-grafted antibody. De-humanizing can includerestoring residues from the donor antibody's framework regions to theCDR grafted antibody, thereby restoring proper folding. Similar“de-humanization” can be achieved by (i) including portions of the“donor” framework region in the “recipient” antibody or (ii) graftingportions of the “donor” antibody framework region into the recipientantibody (along with the grafted donor CDRs).

For a further discussion of antibodies, humanized antibodies, humanengineered, and methods for their preparation, see Kontermann and Dubel,eds., Antibody Engineering, Springer, New York, N.Y., 2001.

Exemplary humanized or human engineered antibodies include IgG, IgM,IgE, IgA, and IgD antibodies. The present antibodies can be of any class(IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa orlambda light chain. For example, a human antibody can comprise an IgGheavy chain or defined fragment, such as at least one of isotypes, IgG1,IgG2, IgG3 or IgG4. As a further example, the present antibodies orfragments can comprise an IgG1 heavy chain and an IgG1 light chain.

The present antibodies and fragments can be human antibodies, such asantibodies which bind IL-1β polypeptides and are encoded by nucleic acidsequences which are naturally occurring somatic variants of humangermline immunoglobulin nucleic acid sequence, and fragments, syntheticvariants, derivatives and fusions thereof. Such antibodies may beproduced by any method known in the art, such as through the use oftransgenic mammals (such as transgenic mice) in which the nativeimmunoglobulin repertoire has been replaced with human V-genes in themammal chromosome. Such mammals appear to carry out VDJ recombinationand somatic hypermutation of the human germline antibody genes in anormal fashion, thus producing high affinity antibodies with completelyhuman sequences.

Human antibodies can also be generated through the in vitro screening ofphage display antibody libraries. See Hoogenboom et al. (1991), J. Mol.Biol. 227: 381; and Marks et al. (1991), J. Mol. Biol. 222: 581. Variousantibody-containing phage display libraries have been described and maybe readily prepared. Libraries may contain a diversity of human antibodysequences, such as human Fab, Fv, and scFv fragments, that may bescreened against an appropriate target. Phage display libraries maycomprise peptides or proteins other than antibodies which may bescreened to identify selective binding agents of IL-1β.

The IL-1β binding antibodies and fragments may comprise one or moreportions that do not bind IL-1β but instead are responsible for otherfunctions, such as circulating half-life, direct cytotoxic effect,detectable labeling, or activation of the recipient's endogenouscomplement cascade or endogenous cellular cytotoxicity. The antibodiesor fragments may comprise all or a portion of the constant region andmay be of any isotype, including IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG(e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In addition to, or instead of,comprising a constant region, antigen-binding compounds of the inventionmay include an epitope tag, a salvage receptor epitope, a label moietyfor diagnostic or purification purposes, or a cytotoxic moiety such as aradionuclide or toxin.

The constant region (when present) of the present antibodies andfragments may be of the γ1, γ2, γ3, γ4, μ, β2, or ε or ε type,preferably of the γ type, more preferably of the y, type, whereas theconstant part of a human light chain may be of the κ or λ type (whichincludes the λ₁, λ₂ and λ₃ subtypes) but is preferably of the κ (type.

Variants also include antibodies or fragments comprising a modified Fcregion, wherein the modified Fc region comprises at least one amino acidmodification relative to a wild-type Fc region. The variant Fc regionmay be designed, relative to a comparable molecule comprising thewild-type Fc region, so as to bind Fc receptors with a greater or lesseraffinity.

For example, the present IL-1β binding antibodies and fragments maycomprise a modified Fc region. Fc region refers to naturally-occurringor synthetic polypeptides homologous to the IgG C-terminal domain thatis produced upon papain digestion of IgG. IgG Fc has a molecular weightof approximately 50 kD. In the present antibodies and fragments, anentire Fc region can be used, or only a half-life enhancing portion. Inaddition, many modifications in amino acid sequence are acceptable, asnative activity is not in all cases necessary or desired.

The Fc region can be mutated, if desired, to inhibit its ability to fixcomplement and bind the Fc receptor with high affinity. For murine IgGFc, substitution of Ala residues for Glu 318, Lys 320, and Lys 322renders the protein unable to direct ADCC. Substitution of Glu for Leu235 inhibits the ability of the protein to bind the Fc receptor withhigh affinity. Various mutations for human IgG also are known (see,e.g., Morrison et al., 1994, The Immunologist 2: 119 124 and Brekke etal., 1994, The Immunologist 2: 125).

In some embodiments, the present an antibodies or fragments are providedwith a modified Fc region where a naturally-occurring Fc region ismodified to increase the half-life of the antibody or fragment in abiological environment, for example, the serum half-life or a half-lifemeasured by an in vitro assay. Methods for altering the original form ofa Fc region of an IgG also are described in U.S. Pat. No. 6,998,253.

In certain embodiments, it may be desirable to modify the antibody orfragment in order to increase its serum half-life, for example, addingmolecules such as PEG or other water soluble polymers, includingpolysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, International Publication No. WO96/32478).Salvage receptor binding epitope refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule.

A salvage receptor binding epitope can include a region wherein any oneor more amino acid residues from one or two loops of a Fc domain aretransferred to an analogous position of the antibody fragment. Even morepreferably, three or more residues from one or two loops of the Fcdomain are transferred. Still more preferred, the epitope is taken fromthe CH2 domain of the Fc region (e.g., of an IgG) and transferred to theCH1, CH3, or VH region, or more than one such region, of the antibody.Alternatively, the epitope is taken from the CH2 domain of the Fc regionand transferred to the CL region or V_(L) region, or both, of theantibody fragment. See also International applications WO 97/34631 andWO 96/32478 which describe Fc variants and their interaction with thesalvage receptor.

Mutation of residues within Fc receptor binding sites can result inaltered effector function, such as altered ADCC or CDC activity, oraltered half-life. Potential mutations include insertion, deletion orsubstitution of one or more residues, including substitution withalanine, a conservative substitution, a non-conservative substitution,or replacement with a corresponding amino acid residue at the sameposition from a different IgG subclass (e.g. replacing an IgG1 residuewith a corresponding IgG2 residue at that position). For example it hasbeen reported that mutating the serine at amino acid position 241 inIgG4 to proline (found at that position in IgG1 and IgG2) led to theproduction of a homogeneous antibody, as well as extending serumhalf-life and improving tissue distribution compared to the originalchimeric IgG4. (Angal et al., Mol. Immunol. 30:105-8, 1993).

Preferably, the antibody or antibody fragment of the present inventiondoes not cross-react with any target other than IL-1β. For example, thepresent antibodies and fragments preferably do not detectably bind toIL-1α.

IL-1β Binding Antibody or Antibody Fragment

Antibody fragments are portions of an intact full length antibody, suchas an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecific,trispecific, and multispecific antibodies (e.g., diabodies, triabodies,tetrabodies); minibodies; chelating recombinant antibodies; tribodies orbibodies; intrabodies; nanobodies; small modular immunopharmaceuticals(SMIP), binding-domain immunoglobulin fusion proteins; camelizedantibodies; V_(HH) containing antibodies; and any other polypeptidesformed from antibody fragments.

The invention provides an IL-1β binding antibody or IL-1β bindingfragment thereof comprising SEQ ID NO: 2. FIG. 1 illustrates the aminoacid sequence of SEQ ID NO: 2. Preferably, the antibody or antibodyfragment comprises the amino acid sequence of SEQ ID NO: 21, and morepreferably comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. Antibodies of theinvention also can comprise the amino acid sequence of SEQ ID NO: 12 orSEQ ID NO: 13, and preferably comprise SEQ ID NO: 14 or SEQ ID NO: 15.Typically, the antibody or antibody fragment comprises a light chainvariable region and a heavy chain variable region, and the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 2 (e.g.,comprises the amino acid sequence of SEQ ID NOS: 4-8, 12-15, or 21). Thelight chain of the antibody preferably comprises, consists essentiallyof, or consists of the amino acid sequence of SEQ ID NO: 1. Thus, forexample, the light chain of the antibody can comprise, consistessentially of, or consist of the amino acid sequence of SEQ ID NO: 9,SEQ ID NO: 10, or SEQ ID NO: 11. Also preferred is an antibody orantibody fragment (e.g., a heavy chain variable region of an antibody orantibody fragment that comprises, consists essentially of, or consistsof the amino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24 (e.g., SEQID NO: 25 or SEQ ID NO: 26).

The invention provides an IL-1β binding antibody or IL-1β bindingfragment thereof comprising a heavy chain variable region comprising oneof the amino acid sequences of SEQ ID NO: 2, 23 or 24, alternatively oneof the amino acid sequence of SEQ ID NO: 12, 13, 21, 23 or 24, oralternatively the amino acid sequence of SEQ ID NO: 12, 13 or 21, oralternatively the amino acid sequence of SEQ ID NO: 13 or 21, oralternatively the amino acid sequence of SEQ ID NO: 8, 14 or 15, oralternatively the amino acid sequence of SEQ ID NO: 8 or 15. Typically,the antibody or antibody fragment comprises a light chain variableregion, preferably comprising of the amino acid sequence of SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. As one example, a preferred antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 8 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO: 11.

The invention also provides an IL-1β binding antibody or IL-1β bindingfragment thereof comprising one of the amino acid sequences of SEQ IDNO: 28. Preferably, the antibody or fragment further comprises one ofthe amino acid sequences of SEQ ID NO: 27.

The invention also provides an IL-1β binding antibody or IL-1β bindingfragment thereof comprising, consists essentially of, or consists of SEQID NO: 29. Preferably, the antibody or antibody fragment comprises,consists essentially of, or consists of the amino acid sequence of SEQID NO: 31-35, or alternatively the amino acid sequence of SEQ ID NO: 31,32 or 33, or alternatively the amino acid sequence of SEQ ID NO: 32 or33. Preferably, the antibody or antibody fragment further comprises alight chain variable region comprising one of the amino acid sequencesof SEQ ID NO: 27, alternatively one of the amino acid sequences of SEQID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

FIGS. 2, 3, and 4 set forth the heavy and light chain variable regionsof exemplary antibodies of the invention, which sequences correspond toantibodies referred to herein as AB1, AB2, AB3, AB4, AB5, AB5.1, AB5.2,AB5.3, and AB5.4. The AB5.1, AB5.2, AB5.3, and AB5.4 sequences containvariable positions, designated as X1 and X2, in the heavy chain CDR3region. These variable positions can be any of the indicated aminoacids. Preferably X1 and X2 are, respectively, alanine and arginine,valine and arginine, phenylalanine and arginine, lysine and lysine, orasparagine and arginine.

AB5.1 comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:12 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:10. AB5.2 comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:13 and alight chain variable region comprising the amino acid sequence of SEQ IDNO:11. AB5.3 comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:23 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:10. AB5.4 comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO:24 and a light chain variable region comprising the amino acidsequence of SEQ ID NO:11.

The present invention encompasses IL-1β binding antibody fragmentscomprising any of the foregoing heavy or light chain sequences and whichbind IL-1. The term fragments as used herein refers to any 3 or morecontiguous amino acids (e.g., 4 or more, 5 or more 6 or more, 8 or more,or even 10 or more contiguous amino acids) of the antibody andencompasses Fab, Fab′, F(ab′)₂, and F(v) fragments, or the individuallight or heavy chain variable regions or portion thereof. IL-1β bindingfragments include, for example, Fab, Fab′, F(ab′)₂, Fv and scFv. Thesefragments lack the Fc fragment of an intact antibody, clear more rapidlyfrom the circulation, and can have less non-specific tissue binding thanan intact antibody. See Wahl et al. (1983), J. Nucl. Med., 24: 316-25.These fragments can be produced from intact antibodies using well knownmethods, for example by proteolytic cleavage with enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments).

The present invention encompasses IL-1β binding antibodies and IL-1βbinding fragments thereof that selectively bind to the IL-1β ligand, butpermit or substantially permit the binding of the bound IL-1β ligand toIL-1β receptor type I (IL-1RI) (see Example 14 and FIGS. 19 and 20). Incontrast to many antibodies, including several known IL-1β bindingantibodies, the antibodies designated AB5 and AB7 selectively bind tothe IL-1β ligand, but they do not block or substantially block thebinding of IL-1β to IL-1RI, as demonstrated in Example 14. For example,the antibody designated AB7 binds to an IL-1β epitope but still permitsthe bound IL-1β to binds to IL-1RI. Thus, the present inventionencompasses IL-1β binding antibodies or fragments that bind to an IL-1βepitope such that the bound antibody or fragment permits orsubstantially permits the IL-1β from binding to IL-1β receptor I(IL-1RI), and the antibody or fragment binds to human IL-1β with adissociation constant less than 3 pM.

In vitro and cell based assays are well described in the art for use indetermining binding of IL-1β to IL-1 receptor type I, including assaysthat determining in the presence of molecules (such as antibodies,antagonists, or other inhibitors) that bind to IL-1β or IL-1RI. (see forexample Evans et al., (1995), J. Biol. Chem. 270:11477-11483; Vigers etal., (2000), J. Biol. Chem. 275:36927-36933; Yanofsky et al., (1996),Proc. Natl. Acad. Sci. USA 93:7381-7386; Fredericks et al., (2004),Protein Eng. Des. Sel. 17:95-106; Slack et al., (1993), J. Biol. Chem.268:2513-2524; Smith et al., (2003), Immunity 18:87-96; Vigers et al.,(1997), Nature 386:190-194; Ruggiero et al., (1997), J. Immunol.158:3881-3887; Guo et al., (1995), J. Biol. Chem. 270:27562-27568;Svenson et al., (1995), Eur. J. Immunol. 25:2842-2850; Arend et al.,(1994), J. Immunol. 153:4766-4774). Recombinant IL-1 receptor type I,including human IL-1 receptor type I, for such assays is readilyavailable from a variety of commercial sources (see for example R&DSystems, SIGMA). IL-1 receptor type I also can be expressed from anexpression construct or vector introduced into an appropriate host cellusing standard molecular biology and transfection techniques known inthe art. The expressed IL-1 receptor type I may then be isolated andpurified for use in binding assays, or alternatively used directly in acell associated form.

For example, the binding of IL-1β to IL-1 receptor type I may bedetermined by immobilizing an IL-1β binding antibody, contacting IL-1βwith the immobilized antibody and determining whether the IL-1β wasbound to the antibody, and contacting a soluble form of IL-1RI with thebound IL-1β/antibody complex and determining whether the soluble IL-1RIwas bound to the complex. The protocol may also include contacting thesoluble IL-1RI with the immobilized antibody before the contact withIL-1β, to confirm that the soluble IL-1RI does not bind to theimmobilized antibody. This protocol can be performed using a Biacore®instrument for kinetic analysis of binding interactions. Such a protocolcan also be employed to determine whether an antibody or other moleculepermits or blocks the binding of IL-1β to IL-1 receptor type I. Forother IL-1β/IL-1RI binding assays, the permitting or blocking of IL-1βbinding to IL-1 receptor type I may be determined by comparing thebinding of IL-1β to IL-1RI in the presence or absence of IL-1βantibodies or IL-1β binding fragments thereof. Blocking is identified inthe assay readout as a designated reduction of IL-1β binding to IL-1receptor type I in the presence of anti-IL-1β antibodies or IL-1βbinding fragments thereof, as compared to a control sample that containsthe corresponding buffer or diluent but not an IL-1β antibody or IL-1βbinding fragment thereof. The assay readout may be qualitatively viewedas indicating the presence or absence of blocking, or may bequantitatively viewed as indicating a percent or fold reduction inbinding due to the presence of the antibody or fragment.

Alternatively or additionally, when an IL-1β binding antibody or IL-1βbinding fragment substantially blocks IL-1β binding to IL1RI, the IL-1βbinding to IL1RI is reduced by at least 10-fold, alternatively at leastabout 20-fold, alternatively at least about 50-fold, alternatively atleast about 100-fold, alternatively at least about 1000-fold,alternatively at least about 10000-fold, or more, compared to binding ofthe same concentrations of IL-1β and IL1RI in the absence of theantibody or fragment. As another example, when an IL-1β binding antibodyor IL-1β binding fragment substantially permits IL-1β binding to IL1RI,the IL-1β binding to IL1RI is at least about 90%, alternatively at leastabout 95%, alternatively at least about 99%, alternatively at leastabout 99.9%, alternatively at least about 99.99%, alternatively at leastabout 99.999%, alternatively at least about 99.9999%, alternativelysubstantially identical to binding of the same concentrations of IL-1βand IL1RI in the absence of the antibody or fragment.

The present invention encompasses IL-1β binding antibodies or IL-1βbinding fragments that bind to the same epitope or substantially thesame epitope as one or more of the exemplary antibodies describedherein. Alternatively or additionally, the IL-1β binding antibodies orIL-1β binding fragments compete with the binding of an antibody havingthe light chain variable region of SEQ ID NO:11 and the heavy chainvariable region of SEQ ID NO:15. Alternatively or additionally, thepresent invention encompasses IL-1β binding antibodies and fragmentsthat bind to an epitope contained in the amino acid sequenceESVDPKNYPKKKMEKRFVFNKIE (SEQ ID NO:36), an epitope that the antibodiesdesignated AB5 and AB7 bind to. As contemplated herein, one can readilydetermine if an IL-1β binding antibody or fragment binds to the sameepitope or substantially the same epitope as one or more of theexemplary antibodies, such as for example the antibody designated AB7,using any of several known methods in the art.

For example, the key amino acid residues (epitope) bound by an IL-1βbinding antibody or fragment may be determined using a peptide arraysimilar to the method described in Example 11. A peptide array, such asfor example, a PepSpot™ peptide array (JPT Peptide Technologies, Berlin,Germany), wherein a scan of twelve amino-acid peptides, spanning theentire IL-1β amino acid sequence, each peptide overlapping by 11 aminoacid to the previous one, is synthesized directly on a membrane. Themembrane carrying the peptides is then probed with the antibody forwhich epitope binding information is sought, for example at aconcentration of 2 μg/ml, for 2 hr at room temperature. Binding ofantibody to membrane bound peptides may be detected using a secondaryHRP-conjugated goat anti-human (or mouse, when appropriate) antibody,followed by enhanced chemiluminescence (ECL). The peptides spot(s)corresponding to particular amino acid residues or sequences of themature IL-1β protein, and which score positive for antibody binding, areindicative of the epitope bound by the particular antibody.

Alternatively or in addition, antibody competition experiments may beperformed and such assays are well known in the art. For example, todetermine if an antibody or fragment binds to an epitope contained in apeptide sequence comprising the amino acids ESVDPKNYPKKKMEKRFVFNKIE,which corresponds to residues 83-105 of the mature IL-1β protein, anantibody of unknown specificity may be compared with any of theexemplary of antibodies (e.g., AB7) of the present invention that areknown to bind an epitope contained within this sequence. Bindingcompetition assays may be performed, for example, using a Biacore®instrument for kinetic analysis of binding interactions or by ELISA. Insuch an assay, the antibody of unknown epitope specificity is evaluatedfor its ability to compete for binding against the known comparatorantibody (e.g., AB7). Competition for binding to a particular epitope isdetermined by a reduction in binding to the IL-1β epitope of at leastabout 50%, or at least about 70%, or at least about 80%, or at leastabout 90%, or at least about 95%, or at least about 99% or about 100%for the known comparator antibody (e.g., AB7) and is indicative ofbinding to substantially the same epitope.

In view of the identification in this disclosure of IL-1β bindingregions in exemplary antibodies and/or epitopes recognized by thedisclosed antibodies, it is contemplated that additional antibodies withsimilar binding characteristics and therapeutic or diagnostic utilitycan be generated that parallel the embodiments of this disclosure.

Furthermore, the IL-1β antibodies and fragments of the present inventionencompass any of the foregoing amino acid sequences of the light orheavy chains with one or more conservative substitutions (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or conservative substitutions). Inlight of the present disclosure, one can determine the positions of anamino acid sequence that are candidates for conservative substitutions,and one can select synthetic and naturally-occurring amino acids thateffect conservative substitutions for any particular amino acids.Consideration for selecting conservative substitutions include thecontext in which any particular amino acid substitution is made, thehydrophobicity or polarity of the side-chain, the general size of theside chain, and the pK value of side-chains with acidic or basiccharacter under physiological conditions. For example, lysine, arginine,and histidine are often suitably substituted for each other. As is knownin the art, this is because all three amino acids have basic sidechains, whereas the pK value for the side-chains of lysine and arginineare much closer to each other (about 10 and 12) than to histidine (about6). Similarly, glycine, alanine, valine, leucine, and isoleucine areoften suitably substituted for each other, with the proviso that glycineis frequently not suitably substituted for the other members of thegroup. This is because each of these amino acids are relativelyhydrophobic when incorporated into a polypeptide, but glycine's lack ofan α-carbon allows the phi and psi angles of rotation (around theα-carbon) so much conformational freedom that glycinyl residues cantrigger changes in conformation or secondary structure that do not oftenoccur when the other amino acids are substituted for each other. Othergroups of amino acids frequently suitably substituted for each otherinclude, but are not limited to, the group consisting of glutamic andaspartic acids; the group consisting of phenylalanine, tyrosine, andtryptophan; and the group consisting of serine, threonine, and,optionally, tyrosine.

By making conservative modifications to the amino acid sequence orcorresponding modifications to the encoding nucleotides, one can produceIL-1β binding antibodies or IL-1β binding fragments having functionaland chemical characteristics similar to those of the exemplaryantibodies and fragments disclosed herein. In contrast, substantialmodifications in the functional and/or chemical characteristics of IL-1βbinding antibodies or IL-1β binding fragments may be accomplished byselecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the molecular backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain.

Antigen-binding fragments of an antibody include fragments that retainthe ability to specifically bind to an antigen, generally by retainingthe antigen-binding portion of the antibody. It is well established thatthe antigen-binding function of an antibody can be performed byfragments of a full-length antibody. Examples of antigen-bindingportions include (i) a Fab fragment, which is a monovalent fragmentconsisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)² fragment,which is a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment which is theVH and CH1 domains; (iv) a Fv fragment which is the VL and VH domains ofa single arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which is a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso encompassed within the term antigen-binding portion of an antibody.The IL-1β binding antibodies and fragments of the present invention alsoencompass monovalent or multivalent, or monomeric or multimeric (e.g.tetrameric), CDR-derived binding domains with or without a scaffold (forexample, protein or carbohydrate scaffolding).

The present IL-1β binding antibodies or fragments may be part of alarger immunoadhesion molecules, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).Antibodies and fragments comprising immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.Preferred antigen binding portions are complete domains or pairs ofcomplete domains.

The IL-1β binding antibodies and fragments of the present invention alsoencompass domain antibody (dAb) fragments (Ward et al., Nature341:544-546, 1989) which consist of a V_(H) domain. The IL-1β bindingantibodies and fragments of the present invention also encompassdiabodies are bivalent antibodies in which V_(H) and V_(L) domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g., EP404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

The IL-1β binding antibodies and fragments of the present invention alsoencompass single-chain antibody fragments (scFv) that bind to IL-1β. AnscFv comprises an antibody heavy chain variable region (V_(H)) operablylinked to an antibody light chain variable region (V_(L)) wherein theheavy chain variable region and the light chain variable region,together or individually, form a binding site that binds IL-1β. An scFvmay comprise a V_(H) region at the amino-terminal end and a V_(L) regionat the carboxy-terminal end. Alternatively, scFv may comprise a V_(L)region at the amino-terminal end and a V_(H) region at thecarboxy-terminal end. Furthermore, although the two domains of the Fvfragment, VL and VH, are coded for by separate genes, they can bejoined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules (known as single chain Fv (scFv); seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883).

An scFv may optionally further comprise a polypeptide linker between theheavy chain variable region and the light chain variable region. Suchpolypeptide linkers generally comprise between 1 and 50 amino acids,alternatively between 3 and 12 amino acids, alternatively 2 amino acids.An example of a linker peptide for linking heavy and light chains in anscFv comprises the 5 amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ IDNO:37). Other examples comprise one or more tandem repeats of thissequence (for example, a polypeptide comprising two to four repeats ofGly-Gly-Gly-Gly-Ser (SEQ ID NO:37)) to create linkers.

The IL-1β binding antibodies and fragments of the present invention alsoencompass heavy chain antibodies (HCAb). Exceptions to the H₂L₂structure of conventional antibodies occur in some isotypes of theimmunoglobulins found in camelids (camels, dromedaries and Ilamas;Hamers-Casterman et al., 1993 Nature 363: 446; Nguyen et al., 1998 J.Mol. Biol. 275: 413), wobbegong sharks (Nuttall et al., Mol. Immunol.38:313-26, 2001), nurse sharks (Greenberg et al., Nature 374:168-73,1995; Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95: 11804), and in thespotted ratfish (Nguyen, et al., “Heavy-chain antibodies in Camelidae; acase of evolutionary innovation,” 2002 Immunogenetics 54(1): 39-47).These antibodies can apparently form antigen-binding regions using onlyheavy chain variable region, in that these functional antibodies aredimers of heavy chains only (referred to as “heavy-chain antibodies” or“HCAbs”). Accordingly, some embodiments of the present IL-1β bindingantibodies and fragments may be heavy chain antibodies (HCAb) thatspecifically bind to IL-1. For example, heavy chain antibodies that area class of IgG and devoid of light chains are produced by animals of thegenus Camelidae which includes camels, dromedaries and llamas(Hamers-Casterman et al., Nature 363:446-448 (1993)). HCAbs have amolecular weight of about 95 kDa instead of the about 160 kDa molecularweight of conventional IgG antibodies. Their binding domains consistonly of the heavy-chain variable domains, often referred to as V_(HH) todistinguish them from conventional V_(H). Muyldermans et al., J. Mol.Recognit. 12:131-140 (1999). The variable domain of the heavy-chainantibodies is sometimes referred to as a nanobody (Cortez-Retamozo etal., Cancer Research 64:2853-57, 2004). A nanobody library may begenerated from an immunized dromedary as described in Conrath et al.,(Antimicrob Agents Chemother 45: 2807-12, 2001) or using recombinantmethods.

Since the first constant domain (C_(H1)) is absent (spliced out duringmRNA processing due to loss of a splice consensus signal), the variabledomain (V_(HH)) is immediately followed by the hinge region, the C_(H2)and the C_(H3) domains (Nguyen et al., Mol. Immunol. 36:515-524 (1999);Woolven et al., Immunogenetics 50:98-101 (1999)). Camelid V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains.

Although the HCAbs are devoid of light chains, they have anantigen-binding repertoire. The genetic generation mechanism of HCAbs isreviewed in Nguyen et al. Adv. Immunol 79:261-296 (2001) and Nguyen etal., Immunogenetics 54:39-47 (2002). Sharks, including the nurse shark,display similar antigen receptor-containing single monomeric V-domains.Irving et al., J. Immunol. Methods 248:31-45 (2001); Roux et al., Proc.Natl. Acad. Sci. USA 95:11804 (1998).

V_(HH)s comprise small intact antigen-binding fragments (for example,fragments that are about 15 kDa, 118-136 residues). Camelid V_(HH)domains have been found to bind to antigen with high affinity (Desmyteret al., J. Biol. Chem. 276:26285-90, 2001), with V_(HH) affinitiestypically in the nanomolar range and comparable with those of Fab andscFv fragments. V_(HH)s are highly soluble and more stable than thecorresponding derivatives of scFv and Fab fragments. V_(H) fragmentshave been relatively difficult to produce in soluble form, butimprovements in solubility and specific binding can be obtained whenframework residues are altered to be more V_(HH)-like. (See, forexample, Reichman et al., J. Immunol Methods 1999, 231:25-38.) V_(HH)scarry amino acid substitutions that make them more hydrophilic andprevent prolonged interaction with BiP (Immunoglobulin heavy-chainbinding protein), which normally binds to the H-chain in the EndoplasmicReticulum (ER) during folding and assembly, until it is displaced by theL-chain. Because of the V_(HH)s′ increased hydrophilicity, secretionfrom the ER is improved.

Functional V_(HH)s may be obtained by proteolytic cleavage of HCAb of animmunized camelid, by direct cloning of V_(HH) genes from B-cells of animmunized camelid resulting in recombinant V_(HH)s, or from naive orsynthetic libraries. V_(HH)s with desired antigen specificity may alsobe obtained through phage display methodology. Using V_(HH)s in phagedisplay is much simpler and more efficient compared to Fabs or scFvs,since only one domain needs to be cloned and expressed to obtain afunctional antigen-binding fragment. Muyldermans, Biotechnol. 74:277-302(2001); Ghahroudi et al., FEBS Lett. 414:521-526 (1997); and van derLinden et al., J. Biotechnol. 80:261-270 (2000). Methods for generatingantibodies having camelid heavy chains are also described in U.S. PatentPublication Nos. 20050136049 and 20050037421.

Ribosome display methods may be used to identify and isolate scFv and/orV_(HH) molecules having the desired binding activity and affinity.Irving et al., J. Immunol. Methods 248:31-45 (2001). Ribosome displayand selection has the potential to generate and display large libraries(10¹⁴).

Other embodiments provide V_(HH)-like molecules generated through theprocess of camelisation, by modifying non-Camelidae V_(H)s, such ashuman V_(HH)s, to improve their solubility and prevent non-specificbinding. This is achieved by replacing residues on the V_(L)s side ofV_(H)s with V_(HH)-like residues, thereby mimicking the more solubleV_(HH) fragments. Camelised V_(H) fragments, particularly those based onthe human framework, are expected to exhibit a greatly reduced immuneresponse when administered in vivo to a patient and, accordingly, areexpected to have significant advantages for therapeutic applications.Davies et al., FEBS Lett. 339:285-290 (1994); Davies et al., ProteinEng. 9:531-537 (1996); Tanha et al., J. Biol. Chem. 276:24774-24780(2001); and Riechmann et al., Immunol. Methods 231:25-38 (1999).

A wide variety of expression systems are available for the production ofIL-1β fragments including Fab fragments, scFv, and V_(HH)s. For example,expression systems of both prokaryotic and eukaryotic origin may be usedfor the large-scale production of antibody fragments and antibody fusionproteins. Particularly advantageous are expression systems that permitthe secretion of large amounts of antibody fragments into the culturemedium.

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786: 161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab. A “minibody”consisting of scFv fused to CH₃ via a peptide linker (hingeless) or viaan IgG hinge has been described in Olafsen, et al., Protein Eng Des Sel.2004 April; 17(4):315-23.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA. 101:17616-21, 2004). Intrabodies, which comprise cell signal sequences whichretain the antibody construct in intracellular regions, may be producedas described in Mhashilkar et al (EMBO J. 14:1542-51, 1995) and Wheeleret al. (FASEB J. 17:1733-5. 2003). Transbodies are cell-permeableantibodies in which a protein transduction domains (PTD) is fused withsingle chain variable fragment (scFv) antibodies Heng et al., (Med.Hypotheses. 64:1105-8, 2005).

The IL-1β binding antibodies and fragments of the present invention alsoencompass antibodies that are SMIPs or binding domain immunoglobulinfusion proteins specific for target protein. These constructs aresingle-chain polypeptides comprising antigen binding domains fused toimmunoglobulin domains necessary to carry out antibody effectorfunctions. See e.g., WO03/041600, U.S. Patent publication 20030133939and US Patent Publication 20030118592.

The IL-1β binding antibodies and fragments of the present invention alsoencompass immunoadhesins. One or more CDRs may be incorporated into amolecule either covalently or noncovalently to make it an immunoadhesin.An immunoadhesin may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs disclosedherein permit the immunoadhesin to specifically bind to IL-1β.

The IL-1β binding antibodies and fragments of the present invention alsoencompass antibody mimics comprising one or more IL-1β binding portionsbuilt on an organic or molecular scaffold (such as a protein orcarbohydrate scaffold). Proteins having relatively definedthree-dimensional structures, commonly referred to as protein scaffolds,may be used as reagents for the design of antibody mimics. Thesescaffolds typically contain one or more regions which are amenable tospecific or random sequence variation, and such sequence randomizationis often carried out to produce libraries of proteins from which desiredproducts may be selected. For example, an antibody mimic can comprise achimeric non-immunoglobulin binding polypeptide having animmunoglobulin-like domain containing scaffold having two or moresolvent exposed loops containing a different CDR from a parent antibodyinserted into each of the loops and exhibiting selective bindingactivity toward a ligand bound by the parent antibody.Non-immunoglobulin protein scaffolds have been proposed for obtainingproteins with novel binding properties. (Tramontano et al., J. Mol.Recognit. 7:9, 1994; McConnell and Hoess, J. Mol. Biol. 250:460, 1995).Other proteins have been tested as frameworks and have been used todisplay randomized residues on alpha helical surfaces (Nord et al., Nat.Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loopsbetween alpha helices in alpha helix bundles (Ku and Schultz, Proc.Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfidebridges, such as those of the small protease inhibitors (Markland etal., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058,1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol.Chem. 270:12250, 1995). Methods for employing scaffolds for antibodymimics are disclosed in U.S. Pat. No. 5,770,380 and US PatentPublications 2004/0171116, 2004/0266993, and 2005/0038229.

Thus, a variety of IL-1β binding antibodies and fragments comprisingone, two, and/or three CDRs of a heavy chain variable region or a lightchain variable region of an antibody (preferably one or more of the CDRsof SEQ ID NOS: 1-26) may be generated.

Preferred antibodies or fragments of the present invention bind to IL-1βwith (i) an IC₅₀ of about 0.5 nM or less (e.g., about 0.4 or less, about0.3 or less, or even about 0.2 or less), as determined by enzyme linkedimmunosorbent assay (ELISA), (ii) at least about 100 times (e.g., atleast about 150 times, at least about 200 times, or even at least about250 times) greater affinity relative to its binding of IL-1α (i.e., hasa selectivity for IL-1β over IL-1β of at least about 100 times (e.g., atleast about 150 times, at least about 200 times, or even at least about250 times)), and/or (iii) an equilibrium binding dissociation constant(K_(D)) for IL-1β of about 20 pM or less (e.g., about 15 pM or less,about 10 pM or less, or even about 5 pM or less). Also preferred areantibodies or fragments of the invention that can inhibit IL-1β inducedexpression of serum IL-6 in an animal by at least 50% (e.g., at least60%, at least 70%, or even at least 80%) as compared to the level ofserum IL-6 in an IL-1β stimulated animal that has not been administeredan antibody or fragment of the invention. Accordingly, the inventionprovides, in a related aspect, an IL-1β binding antibody or IL-1βbinding antibody fragment that has at least one of the aforementionedcharacteristics.

Although the invention has been described herein with respect to IL-1βbinding antibodies and fragments thereof (e.g., comprising a light andheavy chain), the invention also provides polypeptides other than IL-1βbinding antibodies or antibody fragments, such as single-chainpolypeptides (including fusion polypeptides, chimeric polypeptides,conjugates and the like). Thus, the invention provides, in this regard,a polypeptide comprising an amino acid sequence of any of SEQ ID NOS:1-26, or a functionally equivalent fragment or variant thereof. Theinvention also provides a polypeptide comprising an amino acid sequenceof any of SEQ ID NOS: 27-35 or 42-57, or a functionally equivalentfragment or variant thereof.

The antibodies and antibody fragments described herein can be preparedby any suitable method. Suitable methods for preparing such antibodiesand antibody fragments are known in the art. Other methods for preparingthe antibodies and antibody fragments are as described herein as part ofthe invention. The antibody, antibody fragment, or polypeptide of theinvention, as described herein, can be isolated or purified to anydegree. As used herein, an isolated compound is a compound that has beenremoved from its natural environment. A purified compound is a compoundthat has been increased in purity, such that the compound exists in aform that is more pure than it exists (i) in its natural environment or(ii) when initially synthesized and/or amplified under laboratoryconditions, wherein “purity” is a relative term and does not necessarilymean “absolute purity.”

Any of the foregoing antibodies, antibody fragments, or polypeptides ofthe invention can be humanized or human engineered, as described herein.

Methods of Preparing IL-1β Antibodies or Fragments

The invention provides a method of preparing an affinity matured IL-1βbinding polypeptide, such as an antibody or antibody fragment (includingan antibody region (e.g., a light or heavy chain variable region or anypart thereof, such as a CDR)), which method comprises (a) providing afirst nucleic acid comprising a nucleic acid sequence encoding an IL-1βbinding polypeptide that comprises the amino acid sequence of any of SEQID NOs: 1-26 and a second nucleic acid comprising a nucleic acidsequence that differs from the first nucleic acid sequence by at leastone nucleotide, (b) performing nucleic acid shuffling to provide two ormore mutated nucleic acids, and (c) selecting for a mutated nucleic acidthat encodes a polypeptide that either (i) binds to IL-1β with a greateraffinity than the polypeptide encoded by the first nucleic acid, (ii)has a selectivity for IL-1β over IL-1α that is greater than that of thepolypeptide encoded by the first nucleic acid, (iii) has an equilibriumbinding dissociation constant (K_(D)) for IL-1β that is lower than thatof the polypeptide encoded by the first nucleic acid, or (iv) inhibitsIL-1β induced expression of serum IL-6 in an animal to a greater degreethan the polypeptide encoded by the first nucleic acid, and (d)expressing the selected mutated nucleic acid to provide an affinitymatured IL-1β polypeptide. Preferably, the polypeptide is an antibody orantibody fragment, such as any antibody or antibody fragment describedherein as part of the invention.

The method of preparing an affinity matured IL-1β polypeptide optionallyfurther comprises repeating steps (b) and (c) one or more times, whereinthe nucleic acid shuffling of step (b) is performed using (i) at leastone selected mutated nucleic acid of step (c) and (ii) at least onenucleic acid having a nucleic acid sequence that differs from theselected mutated nucleic acid by at least one nucleotide. Preferably,steps (b) and (c) are repeated until an optimized nucleic acid isselected. An optimized nucleic acid is selected when it is no longerpossible to select a nucleic acid encoding a polypeptide that hasbinding characteristics with respect to IL-1β (e.g., characteristics(i)-(iv) of step (c)) that are superior to those of a polypeptideencoded by a nucleic acid previously selected.

Desirably, steps (b) and (c) are repeated until a nucleic acid isselected that encodes a polypeptide having at least one of the followingproperties: (i) binds to IL-1β with an IC₅₀ of about 0.5 nM or less(e.g., about 0.4 or less, about 0.3 or less, or even about 0.2 or less),as determined by enzyme linked immunosorbent assay (ELISA), (ii) bindsto IL-1β with at least about 100 times (e.g., at least about 150 times,at least about 200 times, or even at least about 250 times) greateraffinity relative to its binding of IL-1α (i.e., has a selectivity forIL-1β over IL-1β of at least about 100 times (e.g., at least about 150times, at least about 200 times, or even at least about 250 times)),(iii) binds to IL-1β with an equilibrium binding dissociation constant(K_(D)) for IL-1β of about 20 pM or less (e.g., about 15 pM or less,about 10 pM or less, 5 pM or less, 3 pM or less, 2 pM or less, 1 pm orless, 0.7 pM or less, 0.5 pM or less, 0.3 pM or less, or 0.2 pM orless), or (iv) inhibits IL-1β induced expression of serum IL-6 in ananimal by at least 50% (e.g., at least 60%, at least 70%, or even atleast 80%) as compared to the level of serum IL-6 in an IL-1β stimulatedanimal that has not been administered an antibody or fragment of theinvention.

Selecting for a mutated nucleic acid that encodes a polypeptide havingthe desired properties can be performed by any suitable method.Procedures for expressing encoded polypeptides and assaying thepolypeptides for binding affinity, binding selectivity, equilibriumbinding constants, and inhibition of IL-1β induced IL-6 expression aredisclosed herein (see Examples). Other suitable methods are known in theart. When the polypeptide encoded by the mutated nucleic acid providedby step (b) is not an antibody or whole antibody fragment (e.g., Fab),it may be necessary to provide an antibody comprising the polypeptide inorder to determine whether the polypeptide meets the selection criteria.Thus, the method of preparing an affinity matured IL-1β polypeptide canfurther comprise a step of providing an antibody comprising thepolypeptide encoded by the mutated nucleic acid, wherein the step ofselecting for the mutated nucleic acid encoding a polypeptide having thedesired properties is performed by assaying the antibody.

Nucleic acid shuffling, as used herein, means fragmenting two or morenucleic acid sequences to provide a pool of random nucleic acidfragments and reassembling the fragments to create two or more mutatednucleic acids. In this regard, a mutated nucleic acid is merely anucleic acid that has a nucleic acid sequence that has been changed.Nucleic acid shuffling can be performed by any suitable method. Manysuitable methods are known in the art, such as those described in U.S.Pat. Nos. 6,489,145; 6,773,900; 6,764,835; 6,740,506; 6,713,282;6,713,281; 6,713,279; 6,709,841; 6,696,275; 6,677,115; 6,673,552;6,656,677; 6,605,449; 6,566,050; 6,562,594: 6,555,315; 6,537,776;6,528,249;6,479,258; 6,455,254; 6,440,668; 6,368,798; 6,361,974;6,358,709; 6,352,842; 6,344,328; 6,335,179; 6,280,926; 6,238,884;6,174,673; 6,171,820; 6,168,919; 6,057,103; 6,054,267; 6,030,7796,001,574; 5,965,408; 5,958,672; 5,939,250; 5,763,239; 6,395,547;6,376,246; 6,372,497; 6,368,861; 6,365,408; 6,365,377; 6,358,740;6,358,742; 6,355,484; 6,344,356; 6,337,186; 6,335,160; 6,323,030;6,319,714; 6,319,713; 6,303,344; 6,297,053; 6,291,242; 6,287,861;6,277,638; 6,180,406; 6,165,793; 6,132,970; 6,117,679; 5,834,252;5,830,721; 5,811,238; 5,605,793.

Nucleic Acids

The antibodies, antibody fragments, and polypeptides of the inventioncan be encoded by a single nucleic acid (e.g., a single nucleic acidcomprising nucleotide sequences that encode the light and heavy chainpolypeptides of the antibody), or by two or more separate nucleic acids,each of which encode a different part of the antibody or antibodyfragment. In this regard, the invention provides one or more nucleicacids that encode any of the forgoing antibodies, antibody fragments, orpolypeptides (e.g., any of the foregoing light or heavy chain variableregions).

According to one aspect of the invention, the invention provides anucleic acid that encodes a heavy chain variable region of an antibodyor a portion thereof. Exemplary nucleic acid sequences are provided inSEQ ID NOS: 39 and 40, which respectively encode the heavy chainvariable region of SEQ ID NO: 15, and the light chain variable region ofSEQ ID NO: 11. In this regard, the invention provides a nucleic acidencoding a polypeptide (e.g., a heavy chain variable region of anantibody) comprising the amino acid sequence of SEQ ID NO: 2,alternatively the sequence of SEQ ID NO: 28, and desirably encodes apolypeptide comprising the amino acid sequence of SEQ ID NO: 21 (e.g., apolypeptide comprising the amino acid sequence of any of SEQ ID NOs:4-8). More preferred are nucleic acid sequences encoding a polypeptide(e.g., a heavy chain variable region) comprising the amino acid sequenceof SEQ ID NO: 12 or SEQ ID NO: 13 (e.g., a polypeptide comprising theamino acid sequence of any of SEQ ID NOs: 14-15), or comprising theamino acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24 (e.g., SEQ ID NO:25 or SEQ ID NO: 26).

Alternatively, or in addition, the nucleic acid of the invention cancomprise a nucleic acid sequence that encodes a light chain variableregion of an antibody or a portion thereof. In this regard, theinvention provides a nucleic acid that encodes a polypeptide (e.g., alight chain variable region) comprising the amino acid sequence of SEQID NO: 1. For example, the nucleic acid sequence can encode apolypeptide comprising the amino acid sequence of SEQ ID NO: 9. Morepreferably, the nucleic acid encodes a polypeptide (e.g., a light chainvariable region) that comprises the amino acid sequence of SEQ ID NO:10or 11.

Also encompassed by the invention are nucleic acids encoding any of theforegoing amino acid sequences of the light or heavy chains thatcomprise one or more conservative substitutions (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions), asdiscussed with respect to the antibody and antibody fragment of theinvention, where the antibody or fragment comprising the substitutionhas the same or substantially the same affinity and specificity ofepitope binding as one or more of the exemplary antibodies, fragmentsand sequences disclosed herein.

The nucleic acid sequences can be determined from the amino acidsequences of the antibodies, antibody fragments, and light or heavychain variable regions described herein by any suitable method, such asby converting such amino acid sequences into the corresponding nucleicacid sequences using the genetic code. The nucleic acids encoding thoseamino acid sequences (such as the amino acid sequences described herein)can be prepared (e.g., the nucleic acid sequences isolated orsynthesized) using methods known in the art, such as those described in,for example, Sambrook et al., Molecular Cloning, a Laboratory Manual,3^(rd) edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,2001; Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, New York, N.Y., 1994; andHerdewijn, ed., Oligonucleotide Synthesis. Methods and Applications(Methods in Molecular Biology), Humana Press, Totowa, N.J., 2004. Thenucleic acids described herein can be isolated or purified to anydegree. As used herein, an isolated compound is a compound that has beenremoved from its natural environment. A purified compound is a compoundthat has been increased in purity, such that the compound exists in aform that is more pure than it exists (i) in its natural environment or(ii) when initially synthesized and/or amplified under laboratoryconditions, wherein “purity” is a relative term and does not necessarilymean “absolute purity.”

The nucleic acids can be purified using any of a variety of techniquesincluding, but not limited to preparative gel electrophoresis orisoelectric focusing, affinity, immunoaffinity or ion exchangechromatography, molecular sieve chromatography, chromatofocusing, orhigh pressure liquid chromatography.

Vectors

The nucleic acids described herein can be inserted into vectors, e.g.,nucleic acid expression vectors and/or targeting vectors. Such vectorscan be used in various ways, e.g., for the expression of an IL-1βbinding antibody or antibody fragment in a cell or transgenic animal.Accordingly, the invention provides a vector comprising any one or moreof the nucleic acids of the invention. A “vector” is any molecule orcomposition that has the ability to carry a nucleic acid sequence into asuitable host cell where synthesis of the encoded polypeptide can takeplace. Typically and preferably, a vector is a nucleic acid that hasbeen engineered, using recombinant DNA techniques that are known in theart, to incorporate a desired nucleic acid sequence (e.g., a nucleicacid of the invention). Desirably, the vector is comprised of DNA.Examples of suitable DNA-based gene transfer vectors include plasmidsand viral vectors. Suitable viral vectors include, for instance,parvoviral-based vectors (e.g., adeno-associated virus (AAV)-basedvectors), retroviral vectors, herpes simplex virus (HSV)-based vectors,AAV-adenoviral chimeric vectors, HIV virus-based vectors, andadenovirus-based vectors. Any of these vectors can be prepared usingstandard recombinant DNA techniques described in, e.g., Sambrook et al.,supra, and Ausubel et al., supra. However, vectors that are not based onnucleic acids, such as liposomes, are also known in the art and can beused in connection with the invention. The inventive vector can be basedon a single type of nucleic acid (e.g., a plasmid) or non-nucleic acidmolecule (e.g., a lipid or a polymer). Alternatively, the vector can bea combination of a nucleic acid and a non-nucleic acid (i.e., a“chimeric” vector). For example, a plasmid harboring the nucleic acidcan be formulated with a lipid or a polymer as a delivery vehicle. Sucha vector is referred to herein as a “plasmid-lipid complex” and a“plasmid-polymer” complex, respectively. The inventive gene transfervector can be integrated into the host cell genome or can be present inthe host cell in the form of an episome.

Nucleic acids of the invention can be inserted into immunoglobulinexpression vectors, for example, the vectors described in McLean et al.,Mol. Immunol., 37: 837-45 (2000); Walls et al., Nucleic Acids Res., 21:2921-9 (1993); and Norderhaug et al., J. Immunol. Meth., 204: 77-87(1997).

Vectors are typically selected to be functional in the host cell inwhich the vector will be used (the vector is compatible with the hostcell machinery such that amplification of the gene and/or expression ofthe gene can occur). A nucleic acid molecule encoding an IL-1β bindingantibody or fragment may be amplified/expressed in prokaryotic, yeast,insect (baculovirus systems) and/or eukaryotic host cells. Selection ofthe host cell will depend in part on whether the IL-1β binding antibodyor fragment is to be post-transitionally modified (e.g., glycosylatedand/or phosphorylated). If so, yeast, insect, or mammalian host cellsare preferable. Further information about expression vectors may befound in Meth. Enz. v. 185 (1990; Goeddel, ed.), Academic Press Inc.,San Diego, Calif.

Expression vectors typically contain one or more of the followingcomponents: a promoter, one or more enhancer sequences, an origin ofreplication, a transcriptional termination sequence, a complete intronsequence containing a donor and acceptor splice site, a leader sequencefor secretion, a ribosome binding site, a polyadenylation sequence, apolylinker region for inserting the nucleic acid encoding thepolypeptide to be expressed, and a selectable marker element.

Vector components may be homologous (from the same species and/or strainas the host cell), heterologous (from a species other than the host cellspecies or strain), hybrid (a combination of different sequences frommore than one source), synthetic, or native sequences which normallyfunction to regulate immunoglobulin expression. Sources of vectorcomponents can be any prokaryotic or eukaryotic organism, any vertebrateor invertebrate organism, or any plant, provided that the components arefunctional in, and can be activated by, the host cell machinery.

An origin of replication is selected based upon the type of host cellbeing used for expression. By way of example, the origin of replicationfrom the plasmid pBR322 (Product No. 303-3s, New England Biolabs,Beverly, Mass.) is useful for most Gram-negative bacteria while variousorigins from SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV)or papillomaviruses (such as HPV or BPV) are useful for cloning vectorsin mammalian cells. Generally, the origin of replication component isnot needed for mammalian expression vectors (for example, the SV40origin is often used because it contains the early promoter).

A transcription termination sequence is typically located 3′ of the endof a polypeptide coding regions and serves to terminate transcription.Transcription termination sequences in prokaryotic cells often comprisea G-C rich fragment followed by a poly T sequence. Transcriptiontermination sequences can be cloned from a library, purchasedcommercially as part of a vector, or synthesized using methods fornucleic acid synthesis such as those described above.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes encode proteins that (a) conferresistance to antibiotics or other toxins, e.g., ampicillin,tetracycline, or kanamycin for prokaryotic host cells, (b) complementauxotrophic deficiencies of the cell; or (c) supply critical nutrientsnot available from complex media. Preferred selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. A neomycin resistance gene may also beused for selection in prokaryotic and eukaryotic host cells.

Other selection genes may be used to amplify the gene which will beexpressed. Amplification is a process where genes which are in greaterdemand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of selectable markers for mammalian cellsinclude dihydrofolate reductase (DHFR) and thymidine kinase. Themammalian cell transformants are placed under selection pressure whichonly the transformants are uniquely adapted to survive by virtue of themarker present in the vector. Selection pressure is imposed by culturingthe transformed cells under conditions in which the concentration ofselection agent in the medium is successively changed, thereby leadingto amplification of both the selection gene and the DNA that encodes anIL-1β antibody or fragment. As a result, increased quantities of anantibody are synthesized from the amplified DNA.

A ribosome binding site is generally present for initiating mRNAtranslation. For example, such a site is characterized by aShine-Dalgamo sequence (prokaryotes) or a Kozak sequence (eukaryotes).The element is typically located 3′ to the promoter and 5′ to the codingsequence of the polypeptide to be expressed. The Shine-Dalgarno sequenceis varied but is typically a polypurine (having a high A-G content).Many Shine-Dalgarno sequences have been identified, each of which can bereadily synthesized using methods set forth above and used in aprokaryotic vector.

A leader, or signal, sequence may be used to direct secretion of apolypeptide. A signal sequence may be positioned within or directly atthe 5′ end of a polypeptide coding region. Many signal sequences havebeen identified and may be selected based upon the host cell used forexpression. A signal sequence may be homologous (naturally occurring) orheterologous to a nucleic acid sequence encoding the protein toexpressed (such as antibody or antigen binding fragment). A heterologoussignal sequence selected should be one that is recognized and processed(cleaved by a signal peptidase) by the host cell. For prokaryotic hostcells that do not recognize and process a native antibody signalsequence, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, or heat-stable enterotoxin II leaders. Foryeast secretion, a native antibody signal sequence may be substituted bythe yeast invertase, alpha factor, or acid phosphatase leaders. Inmammalian cell expression the native signal sequence is generallysatisfactory, although other mammalian signal sequences may be suitable.

In most cases, secretion of an antibody or antigen binding fragment froma host cell will result in the removal of the signal peptide from theantibody or fragment. Thus the mature antibody or fragment will lack anyleader or signal sequence.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various presequencesto improve glycosylation or yield. For example, one may alter thepeptidase cleavage site of a signal peptide, or add prosequences, whichalso may affect glycosylation. The final antibody or fragment may have,in the −1 position (relative to the first amino acid of the matureprotein) one or more additional amino acids incident to expression,which may not have been totally removed. For example, the final antibodyor fragment may have one or two amino acid found in the peptidasecleavage site, attached to the N-terminus. Alternatively, use of someenzyme cleavage sites may result in a slightly truncated form of thedesired antibody or fragment, if the enzyme cuts at such area within themature antibody or fragment.

The expression vectors will typically contain a promoter that isrecognized by the host organism and operably linked to a nucleic acidmolecule encoding an IL-1β binding antibody or antigen binding fragment.Either a native or heterologous promoter may be used depending the hostcell used for expression and the yield desired.

Promoters for use with prokaryotic hosts include the beta-lactamase andlactose promoter systems; alkaline phosphatase, a tryptophan (trp)promoter system; and hybrid promoters such as the tac promoter. Otherknown bacterial promoters are also suitable. Their sequences have beenpublished, and they can be ligated to a desired nucleic acidsequence(s), using linkers or adapters as desired to supply restrictionsites.

Promoters for use with yeast hosts are also known in the art. Yeastenhancers are advantageously used with yeast promoters. Suitablepromoters for use with mammalian host cells are well known and includethose obtained from the genomes of viruses such as polyoma virus,fowlpox virus, adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and most preferably Simian Virus 40 (SV40). Other suitablemammalian promoters include heterologous mammalian promoters, e.g.,heat-shock promoters and the actin promoter.

Additional promoters which may be used for expressing the selectivebinding agents of the invention include, but are not limited to: theSV40 early promoter region (Bernoist and Chambon, Nature, 290:304-310,1981); the CMV promoter; the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al. (1980), Cell 22: 787-97);the herpes thymidine kinase promoter (Wagner et al. (1981), Proc. Natl.Acad. Sci. U.S.A. 78: 1444-5); the regulatory sequences of themetallothionine gene (Brinster et al, Nature, 296; 39-42, 1982);prokaryotic expression vectors such as the beta-lactamase promoter(VIIIa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A., 75; 3727-3731,1978); or the tac promoter (DeBoer, et al. (1983), Proc. Natl. Acad.Sci. U.S.A., 80: 21-5). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al. (1984),Cell 38: 639-46; Ornitz et al. (1986), Cold Spring Harbor Symp. Quant.Biol. 50: 399-409; MacDonald (1987), Hepatology 7: 425-515); the insulingene control region which is active in pancreatic beta cells (Hanahan(1985), Nature 315: 115-22); the immunoglobulin gene control regionwhich is active in lymphoid cells (Grosschedl et al. (1984), Cell 38;647-58; Adames et al. (1985), Nature 318; 533-8; Alexander et al.(1987), Mol. Cell. Biol. 7: 1436-44); the mouse mammary tumor viruscontrol region which is active in testicular, breast, lymphoid and mastcells (Leder et al. (1986), Cell 45: 485-95), albumin gene controlregion which is active in liver (Pinkert et al. (1987), Genes and Devel.1: 268-76); the alphafetoprotein gene control region which is active inliver (Krumlauf et al. (1985), Mol. Cell. Biol. 5: 1639-48; Hammer etal. (1987), Science, 235: 53-8); the alpha 1-antitrypsin gene controlregion which is active in the liver (Kelsey et al. (1987), Genes andDevel. 1: 161-71); the beta-globin gene control region which is activein myeloid cells (Mogram et al., Nature, 315 338-340, 1985; Kollias etal. (1986), Cell 46: 89-94); the myelin basic protein gene controlregion which is active in oligodendrocyte cells in the brain (Readheadet al. (1987), Cell, 48: 703-12); the myosin light chain-2 gene controlregion which is active in skeletal muscle (Sani (1985), Nature, 314:283-6); and the gonadotropic releasing hormone gene control region whichis active in the hypothalamus (Mason et al. (1986), Science 234:1372-8).

An enhancer sequence may be inserted into the vector to increasetranscription in eucaryotic host cells. Several enhancer sequencesavailable from mammalian genes are known (e.g., globin, elastase,albumin, alpha-feto-protein and insulin). Typically, however, anenhancer from a virus will be used. The SV40 enhancer, thecytomegalovirus early promoter enhancer, the polyoma enhancer, andadenovirus enhancers are exemplary enhancing elements for the activationof eukaryotic promoters.

While an enhancer may be spliced into the vector at a position 5′ or 3′to the polypeptide coding region, it is typically located at a site 5′from the promoter.

Vectors for expressing nucleic acids include those which are compatiblewith bacterial, insect, and mammalian host cells. Such vectors include,inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen Company, San Diego,Calif.), pBSII (Stratagene Company, La Jolla, Calif.), pET15 (Novagen,Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2(Clontech, Palo Alto, Calif.), pETL (BlueBacII; Invitrogen), pDSR-alpha(PCT Publication No. WO90/14363) and pFastBacDual (Gibco/BRL, GrandIsland, N.Y.).

Additional possible vectors include, but are not limited to, cosmids,plasmids or modified viruses, but the vector system must be compatiblewith the selected host cell. Such vectors include, but are not limitedto plasmids such as Bluescript® plasmid derivatives (a high copy numberColE1-based phagemid, Stratagene Cloning Systems Inc., La Jolla Calif.),PCR cloning plasmids designed for cloning Taq-amplified PCR products(e.g., TOPO™. TA Cloning® Kit, PCR2.1 plasmid derivatives, Invitrogen,Carlsbad, Calif.), and mammalian, yeast or virus vectors such as abaculovirus expression system (pBacPAK plasmid derivatives, Clontech,Palo Alto, Calif.). The recombinant molecules can be introduced intohost cells via transformation, transfection, infection, electroporation,or other known techniques.

Host Cells and Uses Thereof

The invention further provides a cell (e.g., an isolated or purifiedcell) comprising a nucleic acid or vector of the invention. The cell canbe any type of cell capable of being transformed with the nucleic acidor vector of the invention so as to produce a polypeptide encodedthereby. The cell is preferably the cell of a mammal, such as a human,and is more preferably a hybridoma cell, an embryonic stem cell, or afertilized egg.

To express the IL-1β binding or fragment, DNAs encoding partial orfull-length light and heavy chains, obtained as described above, areinserted into expression vectors such that the genes are operativelylinked to transcriptional and translational control sequences. In thiscontext, the term “operatively linked” is intended to mean that anantibody gene is ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the antibodygene. The expression vector and expression control sequences are chosento be compatible with the expression host cell used. The antibody lightchain gene and the antibody heavy chain gene can be inserted intoseparate vector or, more typically, both genes are inserted into thesame expression vector. The antibody genes are inserted into theexpression vector by standard methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present). Prior to insertion of thelight or heavy chain sequences, the expression vector may already carryantibody constant region sequences. For example, one approach toconverting the selected VH and VL sequences to full-length antibodygenes is to insert them into expression vectors already encoding heavychain constant and light chain constant regions, respectively, such thatthe VH segment is operatively linked to the CH segment(s) within thevector and the VL segment is operatively linked to the CL segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention may carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The termregulatory sequence is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, as well as otherconsiderations. Preferred regulatory sequences for mammalian host cellexpression include viral elements that direct high levels of proteinexpression in mammalian cells.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cellswith methotrexate selection/amplification) and the neo gene (for G418selection).

Methods of introducing nucleic acids and vectors into isolated cells andthe culture and selection of transformed host cells in vitro are knownin the art and include the use of calcium chloride-mediatedtransformation, transduction, conjugation, triparental mating, DEAE,dextran-mediated transfection, infection, membrane fusion withliposomes, high velocity bombardment with DNA-coated microprojectiles,direct microinjection into single cells, and electroporation (see, e.g.,Sambrook et al., supra; Davis et al., Basic Methods in MolecularBiology, 2^(nd) ed., McGraw-Hill Professional, 1995; and Neumann et al.,EMBO J., 1: 841 (1982)).

The cell comprising the nucleic acid or vector of the invention can beused to produce the IL-1β binding antibody, fragment thereof, or aportion thereof (e.g., a heavy chain sequence, or a light chain sequenceencoded by the nucleic acid or vector). After introducing the nucleicacid or vector of the invention into the cell, the cell is culturedunder conditions suitable for expression of the encoded sequence. Theantibody, antigen binding fragment, or portion of the antibody then canbe isolated from the cell.

In certain embodiments, two or more vectors that together encode anIL-1β binding antibody, or antigen binding fragment thereof, can beintroduced into the cell. For example, a first vector encoding a heavychain variable region or a complete heavy chain sequence can beintroduced to a host cell, and a second vector encoding a light chainvariable region or complete light chain sequence also is introduced tothe host cell. The cell is then cultured under conditions suitable forexpression of the two sequences encoded by the first and second vectors,and the encoded polypeptides can be isolated from the host cell. Ifnecessary, the isolated polypeptides then can be combined underconditions that promote their association and organization into an IL-1βbinding antibody or antigen binding fragment thereof. Alternatively, thefirst and second vectors can be introduced into separate cells, and theproducts can be isolated from the respective cells and combined toprovide an IL-1β binding antibody or antigen binding fragment thereof.Methods for promoting the association and organization of antibodyconstituents into antigen-binding polypeptides have been described inthe art. Similarly, methods for isolating an antibody, antigen bindingfragment thereof, or heavy chain and light chain fragments are known toordinarily skilled artisans.

Embryonic stem cells or fertilized eggs that comprise a nucleic acid orvector of the invention can be used to generate a transgenic non-humananimal. Methods for making transgenic animals are described in Hofker etal., Transgenic Mouse. Methods and Portocols (Methods in MolecularBiology), Humana Press, Clifton, N.J., 2002. Transgenic non-humananimals that comprise a nucleic acid or vector disclosed herein can beused to express the encoded antibody, antigen binding fragment, orportion of the antibody. The antibody, antigen binding fragment, orportion then can be isolated from the animal. Portions of an antibodycan subsequently be reconstituted (in combination with additionalantibody portions) into an IL-1β binding antibody or antibody fragmentof the invention.

The host cells may be prokaryotic host cells (such as E. coli) oreukaryotic host cells (such as a yeast cell, an insect cell, or avertebrate cell). The host cell, when cultured under appropriateconditions, expresses an IL-1β binding antibody or fragment which cansubsequently be collected from the culture medium (if the host cellsecretes it into the medium) or directly from the host cell producing it(if it is not secreted). Selection of an appropriate host cell willdepend upon various factors, such as desired expression levels,polypeptide modifications that are desirable or necessary for activity,such as glycosylation or phosphorylation, and ease of folding into abiologically active molecule. A number of suitable host cells are knownin the art and many are available from the American Type CultureCollection (ATCC), Manassas, Va. Examples include mammalian cells, suchas Chinese hamster ovary cells (CHO) (ATCC No. CCL61) CHO DHFR-cells(Urlaub et al. Proc. Natl. Acad. Sci. USA 97, 4216-4220 (1980)), humanembryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), 3T3 cells(ATCC No. CCL92), or PER.C6 cells. The selection of suitable mammalianhost cells and methods for transformation, culture, amplification,screening and product production and purification are known in the art.Other suitable mammalian cell lines, are the monkey COS-1 (ATCC No.CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the CV-1 cell line(ATCC No. CCL70). Further exemplary mammalian host cells include primatecell lines, avian cell lines, and rodent cell lines, includingtransformed cell lines. Normal diploid cells, cell strains derived fromin vitro culture of primary tissue, as well as primary explants, arealso suitable. Candidate cells may be genotypically deficient in theselection gene, or may contain a dominantly acting selection gene. Othersuitable mammalian cell lines include but are not limited to, mouseneuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, which areavailable from the American Type Culture Collection, Manassas, Va.).Each of these cell lines is known by and available to those skilled inthe art of protein expression.

Similarly useful as host cells suitable for the present invention arebacterial cells. For example, the various strains of E. coli (e.g.,HB101, (ATCC No. 33694) DH5α, DH10, and MC1061 (ATCC No. 53338)) arewell-known as host cells in the field of biotechnology. Various strainsof B. subtilis, Pseudomonas spp., other Bacillus spp., Streptomycesspp., and the like may also be employed in this method.

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. Transfection encompasses a wide variety oftechniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is theoretically possible to express the IL-1β bindingantibodies or fragments in either prokaryotic or eukaryotic host cells,expression of the antibodies or fragments in eukaryotic cells, and mostpreferably mammalian host cells, is the most preferred because sucheukaryotic cells, and in particular mammalian cells, are more likelythan prokaryotic cells to assemble and secrete a properly folded andimmunologically active antibody. Mammalian host cells for expressing therecombinant antibodies of the invention include Chinese Hamster Ovary(CHO cells) (including dhfr-CHO cells, described in Urlaub and Chasin,(1980) Proc. Natl. Acad. Sci. USA 77:42164220, used with a DHFRselectable marker, e.g., as described in R. J. Kaufman and P. A. Sharp(1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2cells. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Many strains of yeast cells known in the art are also available as hostcells for expression of the antibodies and fragments. Preferred yeastcells include, for example, Saccharomyces cerivisae. Additionally, wheredesired, insect cell systems may be utilized. Such systems are describedfor example in Kitts et al. (Biotechniques, 14, 810-817 (1993)), Lucklow(Curr. Opin. Biotechnol., 4, 564-572 (1993) and Lucklow et al. (J.Virol., 67, 4566-4579 (1993)). Preferred insect cells are Sf-9 and Hi5(Invitrogen, Carlsbad, Calif.).

Transformation or transfection of a nucleic acid molecule encoding anIL-1β binding antibody or fragment into a selected host cell may beaccomplished by well known methods including calcium chloride methods,electroporation methods, microinjection methods, lipofection methods orthe DEAE-dextran methods. The method selected will in part depend on thetype of host cell to be used. These methods and other suitable methodsare well known, and are set forth, for example, in Sambrook et al.supra.

Transgenic animals can also be used to express glycosylated antibodiesand fragments. For example, one may use a transgenic milk-producinganimal (a cow or goat, for example) and obtain glycosylated bindingagents in the animal milk. Alternatively, one may use plants to produceglycosylated selective binding agents.

Host cells comprising an expression vector encoding an IL-1β bindingantibody or fragment may be cultured using media known in the art. Themedia will usually contain all nutrients necessary for the growth andsurvival of the cells. Examples of media for culturing E. coli cellsinclude Luria Broth (LB) and/or Terrific Broth (TB). Suitable media forculturing eukaryotic cells are RPMI 1640, MEM, DMEM, which may besupplemented with serum and/or growth factors as desired for theparticular cell line being cultured. An exemplary medium for insectcultures is Grace's medium supplemented with yeastolate, lactalbuminhydrolysate, and/or fetal calf serum as necessary.

An antibiotic or other compound useful for selective growth oftransfected or transformed cells may be added as a supplement to themedia. The compound will be chosen based on the selectable markerelement present on the plasmid with which the host cell was transformed.For example, where the selectable marker element is kanamycinresistance, the compound added to the culture medium will be kanamycin.Other compounds for selective growth include ampicillin, tetracyclineand neomycin.

The amount of IL-1β binding antibody or fragment produced by a host cellcan be evaluated using methods known in the art. Such methods include,without limitation, Western blot analysis, SDS-polyacrylamide gelelectrophoresis, non-denaturing gel electrophoresis, HPLC separation,immunoprecipitation, and/or activity assays.

Purification of an IL-1β binding antibody or fragment which has beensecreted into the cell media can be accomplished using a variety oftechniques including affinity, immunoaffinity or ion exchangechromatography, molecular sieve chromatography, preparative gelelectrophoresis or isoelectric focusing, chromatofocusing, and highpressure liquid chromatography. For example, antibodies comprising a Fcregion may be purified by affinity chromatography with Protein A, whichselectively binds the Fc region. Modified forms of an antibody orantigen binding fragment may be prepared with affinity tags, such ashexahistidine or other small peptide such as FLAG (Eastman Kodak Co.,New Haven, Conn.) or myc (Invitrogen) at either its carboxyl or aminoterminus and purified by a one-step affinity column. For example,polyhistidine binds with great affinity and specificity to nickel, thusan affinity column of nickel (such as the Qiagen.®. nickel columns) canbe used for purification of polyhistidine-tagged selective bindingagents. (See for example, Ausubel et al, eds., Current Protocols inMolecular Biology, Section 10.11.8, John Wiley & Sons, New York (1993)).In some instances, more than one purification step may be employed.

IL-1β binding antibodies or fragments which are expressed in procaryotichost cells may be present in soluble form either in the periplasmicspace or in the cytoplasm or in an insoluble form as part ofintracellular inclusion bodies. IL-1β binding antibodies or fragmentscan be extracted from the host cell using any appropriate techniqueknown in the art. For example, the host cells can be lysed to releasethe contents of the periplasm/cytoplasm by French press, homogenization,and/or sonication followed by centrifugation.

Soluble forms of an IL-1β binding antibody or fragment present either inthe cytoplasm or released from the periplasmic space may be furtherpurified using methods known in the art, for example Fab fragments arereleased from the bacterial periplasmic space by osmotic shocktechniques.

If inclusion bodies comprising an antibody or fragment have formed, theycan often bind to the inner and/or outer cellular membranes and thuswill be found primarily in the pellet material after centrifugation. Thepellet material can then be treated at pH extremes or with chaotropicagent such as a detergent, guanidine, guanidine derivatives, urea, orurea derivatives in the presence of a reducing agent such asdithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pHto release, break apart, and solubilize the inclusion bodies. Thesoluble antibody or fragment can then be analyzed using gelelectrophoresis, immunoprecipitation or the like. If it is desired toisolate a solublized antibody or antigen binding fragment isolation maybe accomplished using standard methods such as those set forth below andin Marston et al. (Meth. Enz., 182:264-275 (1990)).

In some cases, an IL-1β binding antibody or fragment may not bebiologically active upon isolation. Various methods for “refolding” orconverting a polypeptide to its tertiary structure and generatingdisulfide linkages, can be used to restore biological activity. Suchmethods include exposing the solubilized polypeptide to a pH usuallyabove 7 and in the presence of a particular concentration of achaotrope. The selection of chaotrope is very similar to the choicesused for inclusion body solubilization, but usually the chaotrope isused at a lower concentration and is not necessarily the same aschaotropes used for the solubilization. In most cases therefolding/oxidation solution will also contain a reducing agent or thereducing agent plus its oxidized form in a specific ratio to generate aparticular redox potential allowing for disulfide shuffling to occur inthe formation of the protein's cysteine bridge(s). Some of the commonlyused redox couples include cysteine/cystamine, glutathione(GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT,and 2-mercaptoethanol(bME)/di- thio-b(ME). In many instances, acosolvent may be used to increase the efficiency of the refolding, andcommon reagents used for this purpose include glycerol, polyethyleneglycol of various molecular weights, arginine and the like.

IL-1β binding antibodies or fragments of the present invention may alsobe prepared by chemical synthesis methods (such as solid phase peptidesynthesis) using techniques known in the art such as those set forth byMerrifield et al. (1963), J. Am. Chem. Soc., 85: 2149, Houghten et al.(1985), Proc Natl Acad. Sci. USA, 82: 5132, and Stewart and Young(1984), Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford,Ill. Such antibodies or fragments may be synthesized with or without amethionine on the amino terminus. Chemically synthesized antibodies andantigen binding fragments may be oxidized using methods set forth inthese references to form disulfide bridges. Antibodies and fragments soprepared will retain at least one biological activity associated with anative or recombinantly produced IL-1β binding antibody or fragment.

Pharmaceutical Compositions

IL-1β binding antibodies, antibody fragments, nucleic acids, or vectorsof the invention can be formulated in compositions, especiallypharmaceutical compositions. Such compositions comprise atherapeutically or prophylactically effective amount of an IL-1β bindingantibody, antibody fragment, nucleic acid, or vector of the invention inadmixture with a suitable carrier, e.g., a pharmaceutically acceptableagent. Typically, IL-1β binding antibodies, antibody fragments, nucleicacids, or vectors of the invention are sufficiently purified foradministration to an animal before formulation in a pharmaceuticalcomposition.

Pharmaceutically acceptable agents for use in the present pharmaceuticalcompositions include carriers, excipients, diluents, antioxidants,preservatives, coloring, flavoring and diluting agents, emulsifyingagents, suspending agents, solvents, fillers, bulking agents, buffers,delivery vehicles, tonicity agents, cosolvents, wetting agents,complexing agents, buffering agents, antimicrobials, and surfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. The pharmaceutical compositions can includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also can beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers can be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition can be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see for example U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g. polysorbate 20, polysorbate80); poloxamers (e.g. poloxamer 188); poly (ethylene glycol) phenylethers (e.g. Triton); sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g. fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions can be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, or intralesional routes. A parenteral formulationtypically will be a sterile, pyrogen-free, isotonic aqueous solution,optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, anti-oxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980, which is incorporated herein by reference.

Pharmaceutical compositions described herein can be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) and/orincreased stability or half-life in a particular local environment. Thecompositions can include the formulation of IL-1β binding antibodies,antibody fragments, nucleic acids, or vectors of the invention withparticulate preparations of polymeric compounds such as polylactic acid,polyglycolic acid, etc., as well as agents such as a biodegradablematrix, injectable microspheres, microcapsular particles, microcapsules,bioerodible particles beads, liposomes, and implantable delivery devicesthat provide for the controlled or sustained release of the active agentwhich then can be delivered as a depot injection. Techniques forformulating such sustained- or controlled-delivery means are known and avariety of polymers have been developed and used for the controlledrelease and delivery of drugs. Such polymers are typically biodegradableand biocompatible. Polymer hydrogels, including those formed bycomplexation of enantiomeric polymer or polypeptide segments, andhydrogels with temperature or pH sensitive properties, may be desirablefor providing drug depot effect because of the mild and aqueousconditions involved in trapping bioactive protein agents (e.g.,antibodies). See, for example, the description of controlled releaseporous polymeric microparticles for the delivery of pharmaceuticalcompositions in PCT Application Publication WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly (2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon-(rhIFN—), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present invention. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humour of the eye. Esterified derivatives of hyaluronic acidhave been used to produce microspheres for use in delivery that arebiocompatible and biodegrable (see for example, Cortivo et al.,Biomaterials (1991) 12:727-730; European Publication No. 517,565;International Publication No. WO 96/29998; Illum et al., J. ControlledRel. (1994) 29:133-141). Exemplary hyaluronic acid containingcompositions of the present invention comprise a hyaluronic acid esterpolymer in an amount of approximately 0.1% to about 40% (w/w) of anIL-1β binding antibody or fragment to hyaluronic acid polymer.

Both biodegradable and non-biodegradable polymeric matrices can be usedto deliver compositions of the present invention, and such polymericmatrices may comprise natural or synthetic polymers. Biodegradablematrices are preferred. The period of time over which release occurs isbased on selection of the polymer. Typically, release over a periodranging from between a few hours and three to twelve months is mostdesirable. Exemplary synthetic polymers which can be used to form thebiodegradable delivery system include: polymers of lactic acid andglycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terepthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides,polyurethanes and co-polymers thereof, poly(butic acid), poly(valericacid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, polymers of acrylic and methacrylicesters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,cellulose acetate, cellulose propionate, cellulose acetate butyrate,cellulose acetate phthalate, carboxylethyl cellulose, cellulosetriacetate, cellulose sulphate sodium salt, poly(methyl methacrylate),poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate),poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methylacrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethyleneglycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene andpolyvinylpyrrolidone. Exemplary natural polymers include alginate andother polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion. The polymer optionally is in the formof a hydrogel (see for example WO 04/009664, WO 05/087201, Sawhney, etal., Macromolecules, 1993, 26, 581-587,) that can absorb up to about 90%of its weight in water and further, optionally is cross-linked withmulti-valent ions or other polymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324).

Alternatively or additionally, the compositions can be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which an IL-1β binding antibody,antibody fragment, nucleic acid, or vector of the invention has beenabsorbed or encapsulated. Where an implantation device is used, thedevice can be implanted into any suitable tissue or organ, and deliveryof an IL-1β binding antibody, antibody fragment, nucleic acid, or vectorof the invention can be directly through the device via bolus, or viacontinuous administration, or via catheter using continuous infusion.

A pharmaceutical composition comprising an IL-1β binding antibody,antibody fragment, nucleic acid, or vector of the invention can beformulated for inhalation, such as for example, as a dry powder.Inhalation solutions also can be formulated in a liquefied propellantfor aerosol delivery. In yet another formulation, solutions may benebulized. Additional pharmaceutical composition for pulmonaryadministration include, those described, for example, in PCT ApplicationPublication WO 94/20069, which discloses pulmonary delivery ofchemically modified proteins. For pulmonary delivery, the particle sizeshould be suitable for delivery to the distal lung. For example, theparticle size can be from 1 μm to 5 μm; however, larger particles may beused, for example, if each particle is fairly porous.

Certain formulations containing IL-1β binding antibodies, antibodyfragments, nucleic acids, or vectors of the invention can beadministered orally. Formulations administered in this fashion can beformulated with or without those carriers customarily used in thecompounding of solid dosage forms such as tablets and capsules. Forexample, a capsule can be designed to release the active portion of theformulation at the point in the gastrointestinal tract whenbioavailability is maximized and pre-systemic degradation is minimized.Additional agents can be included to facilitate absorption of aselective binding agent. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders also can be employed.

Another preparation can involve an effective quantity of an IL-1βbinding antibody, antibody fragment, nucleic acid, or vector of theinvention in a mixture with non-toxic excipients which are suitable forthe manufacture of tablets. By dissolving the tablets in sterile water,or another appropriate vehicle, solutions can be prepared in unit doseform. Suitable excipients include, but are not limited to, inertdiluents, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Suitable and/or preferred pharmaceutical formulations can be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose can be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages can be ascertained through use of appropriatedose-response data.

Additional formulations will be evident in light of the presentdisclosure, including formulations involving IL-1β binding antibodies,antibody fragments, nucleic acids, or vectors of the invention incombination with one or more other therapeutic agents. For example, insome formulations, an IL-1β binding antibody, antibody fragment, nucleicacid, or vector of the invention is formulated with a second inhibitorof an IL-1 signaling pathway Representative second inhibitors include,but are not limited to, antibodies, antibody fragments, peptides,polypeptides, compounds, nucleic acids, vectors and pharmaceuticalcompositions, such as, for example, those described in U.S. Pat. No.6,899,878, US 2003022869, US 20060094663, US 20050186615, US20030166069, WO/04022718, WO/05084696, WO/05019259. For example, acomposition may comprise an IL-1β binding antibody, antibody fragment,nucleic acid, or vector of the invention in combination with an IL-1βbinding antibody, fragment, or a nucleic acid or vector encoding such anantibody or fragment.

The pharmaceutical compositions can comprise IL-1β binding antibodies orfragments in combination with other active agents. Such combinations arethose useful for their intended purpose. The active agents set forthbelow are illustrative for purposes and not intended to be limited. Thecombinations which are part of this invention can be the presentantibodies and fragments and at least one additional agent selected fromthe lists below. The combination can also include more than oneadditional agent, e.g., two or three additional agents if thecombination is such that the formed composition can perform its intendedfunction.

Active agents or combinations with the present antibodies or fragmentsinclude a non-steroidal anti-inflammatory drug (NSAID) such as aspirin,ibuprofen, and other propionic acid derivatives (alminoprofen,benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen,flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin,pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen),acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac,ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin,and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamicacid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams(isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetylsalicylic acid, sulfasalazine) and the pyrazolones (apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone).Other combinations include cyclooxygenase-2 (COX-2) inhibitors. Otheractive agents for combination include steroids such as prednisolone,prednisone, methylpredniso lone, betamethas one, dexamethas one, orhydrocortisone. Such a combination may be especially advantageous, sinceone or more side-effects of the steroid can be reduced or eveneliminated by tapering the steroid dose required when treating patientsin combination with the present antibodies and fragments.

Additional examples of active agents for combinations with IL-1β bindingantibodies or fragments for rheumatoid arthritis include cytokinesuppressive anti-inflammatory drug(s) (CSAIDs); antibodies to orantagonists of other human cytokines or growth factors, for example,TNF, LT, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II,GM-CSF, FGF, or PDGF. The IL-1β binding antibodies and fragments can becombined with antibodies to cell surface molecules such as CD2, CD3,CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2),CD90, or their ligands including CD 154 (gp39 or CD40L).

Preferred combinations of active agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade; preferredexamples include TNF antagonists like chimeric, humanized or human TNFantibodies, D2E7, cA2 (Remicade™), CDP 571, anti-TNF antibody fragments(e.g., CDP870), and soluble p55 or p75 TNF receptors, derivativesthereof, (p75TNFRIgG (Enbrel™) or p55TNFRIgG (Lenercept), soluble IL-13receptor (sIL-13), and also TNFα converting enzyme (TACE) inhibitors;similarly IL-1 inhibitors (e.g., Interleukin-1-converting enzymeinhibitors, such as Vx740, or IL-1RA etc.) may be effective for the samereason. Other preferred combinations include Interleukin 11, anti-P7sand p-selectin glycoprotein ligand (PSGL). Yet another combination areother key players of the autoimmune response which may act parallel to,dependent on or in concert with IL-1β function.

Active agents for Crohn's disease in which an antibody or an antigenbinding portion can be combined include TNF antagonists, for example,anti-TNF antibodies, D2E7, cA2 (Remicade™), CDP 571, anti-TNF antibodyfragments (e.g., CDP870), TNFR-Ig constructs(p75TNFRIgG (Enbrel™) andp55TNFRIgG (Lenercept)), anti-P7s, p-selectin glycoprotein ligand(PSGL), soluble IL-13 receptor (sIL-13), and PDE4 inhibitors. The IL-1βbinding antibodies or fragments can be combined with corticosteroids,for example, budenoside and dexamethasone. The IL-1β binding antibodiesor fragments may also be combined with agents such as sulfasalazine,5-aminosalicylic acid and olsalazine, and agents which interfere withsynthesis or action of proinflammatory cytokines such as IL-1, forexample, IL-1 converting enzyme inhibitors (e.g., Vx740) and IL-1ra. TheIL-1β binding antibodies or fragments may also be used with T cellsignaling inhibitors, for example, tyrosine kinase inhibitors6-mercaptopurines. The IL-1β binding antibodies or fragments can becombined with IL-11.

Other examples of active agents for multiple sclerosis with which theIL-1β binding antibodies or fragments can be combined includecorticosteroids; prednisolone; methylprednisolone; azathioprine;cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine;tizanidine; interferon-β1a (Avonex; Biogen); interferon-β1b (Betaseron;Chiron/Berlex); Copolymer 1 (Cop-1; Copaxone; Teva PharmaceuticalIndustries, Inc.); hyperbaric oxygen; intravenous immunoglobulin;clabribine; antibodies to or antagonists of other human cytokines orgrowth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. The IL-1β bindingantibodies or fragments can be combined with antibodies to cell surfacemolecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,CD69, CD80, CD86, CD90 or their ligands. The IL-1β binding antibodies orfragments may be combined with agents, such as methotrexate,cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide,NSAIDs, for example, ibuprofen, corticosteroids such as prednisolone,phosphodiesterase inhibitors, adensosine agonists, antithromboticagents, complement inhibitors, adrenergic agents, agents which interferewith signalling by proinflammatory cytokines such as TNFα or IL-1 (e.g.IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1β converting enzymeinhibitors (e.g., Vx740), anti-P7s, p-selectin glycoprotein ligand(PSGL), TACE inhibitors, T-cell signalling inhibitors such as kinaseinhibitors, metalloproteinase inhibitors, sulfasalazine, azathioprine,6-mercaptopurines, angiotensin converting enzyme inhibitors, solublecytokine receptors and derivatives thereof (e.g. soluble p55 or p75 TNFreceptors, sIL-1 RI, sIL-1RII, sIL-6R, soluble IL-13 receptor (sIL-13))and antiinflammatory cytokines (e.g. IL-4, IL-10, IL-13 and TGFβ).

Preferred examples of active agents for multiple sclerosis in which theIL-1β binding antibodies or fragments can be combined to includeinterferon-β, for example, IFNβ1a and IFNβ1b; copaxone, corticosteroids,IL-1 inhibitors, TNF inhibitors, and antibodies to CD40 ligand and CD80.

The pharmaceutical compositions may include a therapeutically effectiveamount or a prophylactically effective amount of the present IL-1βbinding antibodies or fragments. A therapeutically effective amountrefers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result. A therapeuticallyeffective amount of the antibody or antibody portion may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the antibody or antibody portion toelicit a desired response in the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody or antibody portion are outweighed by the therapeuticallybeneficial effects. A prophylactically effective amount refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

IL-1β binding antibodies, antibody fragments, nucleic acids, or vectorsof the invention, can be employed alone or in combination with otheractive agents, which can be in the same pharmaceutical composition or ina different pharmaceutical composition. For example, such other activeagents can comprise (i) IL-1 antagonist (e.g., recombinant IL-1Ra or anIL-trap), (ii) an interleukin-1 receptor antagonist, (iii) a soluble TNFreceptor-1, (iv) a soluble TNF receptor-2 (e.g., etanercept), (iv) TNFinhibitor (e.g., an antibody such as D2E7), and/or (v) a cancer therapyagent. Thus, for example, one or more of these components can beincluded in the composition of the invention with an IL-1β bindingantibody, antibody fragment, nucleic acid, or vector of the invention.

It may be desirable in some instances to use a pharmaceuticalcomposition comprising an IL-1β binding antibody, antibody fragment,nucleic acid or vector of the invention in an ex vivo manner. In thiscase, cells, tissues, or organs that have been removed from a patientare exposed to pharmaceutical compositions comprising an IL-1β bindingantibody, antibody fragment, nucleic acid, or vector of the invention,after which the cells, tissues, and/or organs are subsequently implantedback into the patient.

In certain situations, a composition comprising an IL-1β bindingantibody, antibody fragment, nucleic acid, or vector can be delivered byimplanting into patients cells that have been genetically engineered, asdescribed herein, to express and secrete the polypeptides, selectivebinding agents, fragments, variants, or derivatives. Such cells may beanimal or human cells, and can be derived from the patient's own tissueor from another source, either human or non-human. Optionally, the cellscan be immortalized cells. However, in order to decrease the chance ofan immunological response, it is preferred that the cells beencapsulated to avoid infiltration of surrounding tissues. Theencapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow release of the proteinproduct(s) but prevent destruction of the cells by the patient's immunesystem or by other detrimental factors from the surrounding tissues.

Methods used for membrane encapsulation of cells are known, and thepreparation of encapsulated cells and their implantation in patients hasbeen described, for example, in U.S. Pat. Nos. 4,892,538, 5,011,472, and5,106,627. A system for encapsulating living cells is described in PCTApplication Publication WO 91/10425. Techniques for formulating avariety of other sustained or controlled delivery means, such asliposome carriers, bio-erodible particles, or beads, are also known tothose in the art, and are described. The cells, with or withoutencapsulation, can be implanted into suitable body tissues or organs ofthe patient.

A therapeutically or prophylactically effective amount of apharmaceutical composition comprising an IL-1β binding antibody,antibody fragment, nucleic acid, or vector of the invention will depend,for example, upon the therapeutic objectives such as the indication forwhich the composition is being used, the route of administration, andthe condition of the subject. Pharmaceutical compositions areadministered in a therapeutically or prophylactically effective amountto treat an IL-1 related condition. A “therapeutically orprophylactically effective amount” of an IL-1β binding antibody,antibody fragment, nucleic acid, or vector of the invention is thatamount which can treat or prevent one or more symptoms of an IL-1related disease in a subject.

Accordingly, it may be desirable to titer the dosage of the IL-1 bindingantibody, antibody fragment, nucleic acid, or vector of the inventionand modify the route of administration as required to obtain the optimaltherapeutic effect. Dosage ranges include from about 0.1 ng/kg to up toabout 100 mg/kg or more (in terms of active agent amount per unit ofbody weight of subject administered the active agent), depending on thefactors mentioned above. In other embodiments, the dosage ranges fromabout 0.1 μg/kg to about 100 mg/kg, from about 1 μg/kg to about 100mg/kg, from about 5 μg/kg to about 100 mg/kg, from about 0.5 mg/kg up toabout 100 mg/kg, or from about 1 mg/kg up to about 100 mg/kg. Otherdosages can be appropriate. The composition can be administered as asingle dose, or as two or more doses (which may or may not contain thesame amount of an IL-1β binding antibody, antibody fragment, nucleicacid, or vector of the invention) over time, or as a continuous infusionvia, for example implantation device or catheter.

Methods of Use

The antibodies, antibody fragments, nucleic acids, vectors, cells, andcompositions of the invention (collectively “the compounds andcompositions of the invention”) can be used for any purpose. Forexample, the compounds and compositions of the invention can be used toresearch IL-1 related mechanisms, as well as the diseases and conditionsassociated with IL-1 related mechanisms. However, the compounds andcompositions of the invention are especially useful to treat a subject(e.g., a mammal or a human) in need of treatment for an IL-1 relatedcondition, e.g., an autoimmune or inflammatory disease or disorder.Accordingly, the invention provides a method of treating or preventing adisease in a mammal comprising administering an effective amount of theantibody or antibody fragment, nucleic acid, or vector of the inventionto a mammal in need thereof, whereby the disease is treated or preventedin the mammal. The term “effective amount” refers to the amount of theantibody or antibody fragment, nucleic acid, or vector of the inventionneeded to establish a prophylactic or therapeutic effect. As usedherein, treating a disease or condition is defined as temporarily orpermanently reducing or eliminating the symptoms or progression of adisease or condition. Similarly, preventing a disease or condition meanstemporarily or permanently inhibiting, slowing, or preventing the onsetof a disease or condition (or the symptoms of a disease or condition).

The method of the invention can be used to treat or prevent any IL-1related disease or condition. For example, the present antibodies andfragments are contemplated for use in the prophylaxis and treatment ofIL-1 mediated diseases or medical conditions, e.g. inflammatoryconditions, allergies and allergic conditions, cancers, hypersensitivityreactions, autoimmune diseases, severe infections, and organ or tissuetransplant rejection. IL-1 related conditions include rheumatoidarthritis (RA), osteoarthritis, Crohn's disease, ulcerative colitis(UC), septic shock, chronic obstructive pulmonary disease (COPD),asthma, graft versus host disease, atherosclerosis, adult T cellleukemia, multiple myeloma, multiple sclerosis, stroke, Alzheimer'sdisease. The present antibodies and fragments can also be used to treator prevent Neonatal Onset Multisystem Inflammatory Disorder(NOMID/CINCA), systemic onset juvenile idiopathic arthritis, Stillsdisease, CAPS, or Muckle-Wells syndrome.

In general, a disease or condition can be considered an IL-1β relateddisease or condition if it is associated with elevated levels of IL-1βin bodily fluids or tissue or if cells or tissues taken from the bodyproduce elevated levels of IL-1β in culture.

For example, the present methods can be used to treat or preventNeonatal Onset Multisystem Inflammatory Disorder (NOMID/CINCA), systemiconset juvenile idiopathic arthritis, CIAS1 Associated Periodic Syndromes(CAPS), Stills disease, or Muckle-Wells syndrome.

As another example, the present methods can be used to treat or preventrheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerativecolitis, septic shock, chronic obstructive pulmonary disease, asthma,graft versus host disease, atherosclerosis, adult T cell leukemia,multiple myeloma, multiple sclerosis, stroke or Alzheimer's disease.

As yet another example, the present methods can be used to treat orprevent systemic onset juvenile idiopathic arthritis, rheumatoidarthritis, osteoarthritis, atherosclerosis, or myasthenia gravis.

Other examples of IL-1β related conditions are acute pancreatitis; ALS;cachexia/anorexia, including AIDS-induced cachexia; asthma and otherpulmonary diseases; autoimmune vasculitis; CIAS1 Associated PeriodicSyndromes (CAPS); chronic fatigue syndrome; Clostridium associatedillnesses, including Clostridium-associated diarrhea; coronaryconditions and indications, including congestive heart failure, coronaryrestenosis, myocardial infarction, myocardial dysfunction (e.g., relatedto sepsis), and coronary artery bypass graft; cancers, such as multiplemyeloma and myelogenous (e.g., AML and CML) and other leukemias, as wellas tumor metastasis; diabetes (e.g., insulin diabetes); endometriosis;familial Cold Autoinflammatory Syndrome (FCAS); familial mediterraneanfever (FMF); fever; fibromyalgia; glomerulonephritis; graft versus hostdisease/transplant rejection; hemohorragic shock; hyperalgesia;inflammatory bowel disease; inflammatory conditions of a joint,including psoriatic arthritis (as well as osteoarthritis and rheumatoidarthritis); inflammatory eye disease, as may be associated with, forexample, corneal transplant; ischemia, including cerebral ischemia(e.g., brain injury as a result of trauma, epilepsy, hemorrhage orstroke, each of which may lead to neurodegeneration); Kawasaki'sdisease; learning impairment; lung diseases (e.g., ARDS); myopathies(e.g., muscle protein metabolism, especially in sepsis); neurotoxicity(e.g., as induced by HIV); osteoporosis; pain, including cancer-relatedpain; Parkinson's disease; periodontal disease; pre-term labor;psoriasis; reperfusion injury; side effects from radiation therapy;sleep disturbance; temporal mandibular joint disease; tumor necrosisfactor receptor-associated periodic fever syndrome (TRAPS); uveitis; oran inflammatory condition resulting from strain, sprain, cartilagedamage, trauma, orthopedic surgery, infection or other diseaseprocesses.

The present antibodies and fragments are also contemplated for use inthe treatment recipients of heart, lung, combined heart-lung, liver,kidney, pancreatic, skin or corneal transplants, including allograftrejection or xenograft rejection, or for the prevention ofgraft-versus-host disease, such as following bone marrow transplant, ororgan transplant associated arteriosclerosis.

The present antibodies and fragments are contemplated for use in thetreatment or prevention of autoimmune disease or inflammatoryconditions, in particular inflammatory conditions with an aetiologyincluding an autoimmune component such as arthritis (for examplerheumatoid arthritis, arthritis chronica progrediente and arthritis andrheumatic diseases, including inflammatory conditions and rheumaticdiseases involving bone loss, inflammatory pain, hypersensitivity(including both airways hypersensitivity and dermal hypersensitivity) orallergies. Specific auto-immune diseases for which the presentantibodies and fragments may be employed include autoimmunehaematological disorders (including e.g. hemolytic anaemia, aplasticanaemia, pure red cell anaemia and idiopathic thrombocytopenia),systemic lupus erythematosus, polychondritis, sclerodoma, Wegenergranulomatosis, chronic active hepatitis, myasthenia gravis, psoriasis,Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory boweldisease (including e.g. ulcerative colitis, Crohn's disease andIrritable Bowel Syndrome), endocrine Graves disease, sarcoidosis,multiple sclerosis, primary biliary cirrhosis, juvenile diabetes(diabetes mellitus type I), uveitis (anterior and posterior),keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitiallung fibrosis, psoriatic arthritis or glomerulonephritis (with andwithout nephrotic syndrome, e.g. including idiopathic nephrotic syndromeor minimal change nephropathy).

The present antibodies and fragments are also contemplated for use inthe treatment, prevention, or amelioration of asthma, bronchitis,pneumoconiosis, pulmonary emphysema, and other obstructive orinflammatory diseases of the airways. The antibodies or fragments fortreating undesirable acute and hyperacute inflammatory reactions whichare mediated by IL-1or involve production, especially or the promotionof TNF release by IL-1, e.g. acute infections, for example septic shock(e.g., endotoxic shock and adult respiratory distress syndrome),meningitis, pneumonia; and severe burns; and for the treatment ofcachexia or wasting syndrome associated with morbid TNF release,consequent to infection, cancer, or organ dysfunction, especiallyAIDS-related cachexia, e.g., associated with or consequential to HIVinfection.

The present antibodies and fragments are also contemplated for use intreating diseases of bone metabolism including osteoarthritis,osteoporosis and other inflammatory arthritides, and bone loss ingeneral, including age-related bone loss, and in particular periodontaldisease.

The present antibodies and fragments are also contemplated for use inthe treatment or prevention of CIAS1 Associated Periodic Syndromes(CAPS), including each of Neonatal Onset Multisystem InflammatoryDisorder (NOMID), Muckle-Wells Syndrome (MWS), and Familial ColdAutoinflammatory Syndrome (FCAS). Mutations in the gene CIAS1 are nowrecognized as being responsible for three rare genetic syndromes:Neonatal Onset Multisystem Inflammatory Disorder (NOMID), Muckle-WellsSyndrome (MWS), and Familial Cold Autoinflammatory Syndrome (FCAS).(Hoffman et al. 2001 Naure 29:301-305; Feldmann et al. 2002 Am J HumGenet. 71:198-203; Aksentijevich et al. 2002 Arthritis Rheum46:3340-3348). In aggregate, these conditions are known as “CAPS.” CIAS1encodes a protein called NALP3 that is a component of the“inflammasome”, a subcellular enzyme complex that regulates the activityof caspase 1. Caspase 1 is the enzyme that cleaves the inactive pro-formof the proinflammatory cytokine, IL-1, into its biologically active form(Agostini et al. 2004 supra). Mutations in CIAS1 lead to increasedproduction of IL-1.

The antibody or antibody fragment, nucleic acid, or vector of theinvention is typically administered to the mammal or human as apharmaceutical composition comprising an antibody or antibody fragment,nucleic acid, or vector of the invention and a pharmaceuticallyacceptable carrier. Pharmaceutical compositions suitable for use inconjunction with the method of treating or preventing a disease are aspreviously described herein.

The antibody or antibody fragment, nucleic acid, or vector of theinvention can be administered to the mammal as the sole active agent, orin conjunction with one or more other agents that disrupt IL-1 receptorsignaling. An agent that disrupts IL-1 receptor signaling can be anycompound or composition that inhibits an interaction between IL-1β andIL-1 receptor. For example, agents that disrupt IL-1 receptor signalinginclude antibodies that bind to IL-1β or to the IL-1 receptor,recombinant IL-1Ra (e.g., from Amgen Inc., Thousand Oaks, Calif.), andIL-1 receptor “trap” peptides (e.g., from Regeneron Inc., Tarrytown,N.Y.). When two or more agents that disrupt IL-1 receptor signaling areused, they can be administered together (e.g., in a singlepharmaceutical composition), or they can each be administered separately(e.g., in separate pharmaceutical compositions).

The antibody, fragment, nucleic acid, or vector of the invention can beadministered to a mammal in combination or in conjunction with one ormore other active agents for treating or preventing IL-1 mediatedconditions or diseases are set forth above.

Diagnostic Uses

In addition to therapeutic uses, the present antibodies and fragmentscan be used in diagnostic methods to detect IL-1β (for example, in abiological sample, such as serum or plasma), using a conventionalimmunoassay, such as an enzyme linked immunosorbent assays (ELISA), anradioimmunoassay (RIA) or tissue immunohistochemistry. A method fordetecting IL-1β in a biological sample can comprise the steps ofcontacting a biological sample with one or more of the presentantibodies or fragments and detecting either the antibody or fragmentbound to IL-1β or unbound antibody or fragment, to thereby detect IL-1βin the biological sample. The antibody or fragment can be directly orindirectly labeled with a detectable substance to facilitate detectionof the bound or unbound antibody. Suitable detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase,or acetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Rather than labeling the antibody, IL-1β can be assayed in biologicalfluids by a competition immunoassay utilizing IL-1β standards labeledwith a detectable substance and an unlabeled anti-IL-1β antibody. Inthis assay, the biological sample, the labeled rIL-1β standards and theanti-IL-1β antibody are combined and the amount of labeled IL-1βstandard bound to the unlabeled antibody is determined. The amount ofIL-1β in the biological sample is inversely proportional to the amountof labeled IL-1β standard bound to the anti-IL-1β antibody.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

In the following Examples, reference is made to various antibodies ofthe present invention, including the antibodies designated AB1, AB5, andAB7. As mentioned above, AB1 comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:4 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO:9. AB5comprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:8 and a light chain variable region comprising theamino acid sequence of SEQ ID NO:9. AB7 comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:15 and a lightchain variable region comprising the amino acid sequence of SEQ IDNO:11.

For various comparisons in the following Examples, reference is made toan antibody designated AB-control, a commercially available antibodywith relatively high affinity for IL-1β. AB-control is a murine antibodywhich is believed to have a heavy chain comprising the sequence of SEQID NO:40 and a light chain comprising the sequence of SEQ ID NO:41.These murine sequences are set forth in U.S. Patent ApplicationPublication No. 2003/0026806, at FIGS. 6A and 6B.

In several of the Examples that follow, AB5 and AB7 are shown to haveunexpectedly higher affinity to human IL-1β than AB-control.

Example 1

This example illustrates the binding affinities of certain antibodies ofthe invention to IL-1β.

Antibodies designated AB1 and AB5 were assayed for IL-1β bindingproperties using a KINEXA™ device (from Sapidyne Instruments Inc.,Boise, Id.). The amino acid sequences of the heavy and light chainvariable regions of antibodies AB1 and AB5 are provided in FIGS. 2 and3. A commercially available antibody with relatively high affinity forIL-1β (herein AB-control) was assayed for comparison.

IL-1β binding assay results are summarized in Table 1. K_(D) valuesrepresent the binding dissociation constants for the respectiveantibody-IL-1β complexes. K_(D) was calculated as the ratio of “offrate” (rate of dissociation for the antibody-IL-1β complex) to “on rate”(rate of association for the antibody-IL-1β complex). A lower K_(D) rateis indicative of higher antibody affinity.

TABLE 1 IL-1β Binding Results Antibody K_(D) (pM) AB-control 3.06 AB1(invention) 18.63 AB5 (invention) 0.261

The results of these experiments show that AB1 and AB5 bind IL-1β withhigh affinity. The affinities for IL-1β of the antibodies of theinvention are comparable to, or better than, the binding affinity ofAB-control for IL-1β.

Example 2

This example illustrates the in vitro inhibition of IL-1β usingantibodies of the invention.

The IL-1β inhibitory potencies of AB1 and AB5 antibodies (see Example 1)were evaluated using a bioassay that measures the IL-1β stimulatedrelease of IL-6 from human fibroblasts. As in Example 1, AB-control wasused as a comparative sample. Details of the assay are described inDinarello et al., Current Protocols in Immunology, Ch. 6.2.1-6.2.7, JohnWiley and Sons Inc., Hoboken, N.J., 2000. Briefly, human MRC5 humanfibroblasts from the American Type Culture Collection (ATCC) Manassas,Va. (ATCC # CCL-171) were grown to confluency in multi-well plates.Cells were treated with titrated doses of AB5 antibody. Cells weresubsequently contacted with (i) 100 pg/ml of IL-1β or (ii) 100 pg/ml ofIL-1β and AB1 or AB5 antibody (from Example 1). Negative control cellswere not stimulated with IL-1β. The amounts of IL-6 released in eachgroup of treated cells were measured using an IL-6 ELISA kit from BDPharmingen (Franklin Lakes, N.J.) according to the manufacturer'sinstructions. ELISA results are depicted in FIG. 5 and summarized inTable 2. IC₅₀ is the concentration of antibody required to inhibit 50%of IL-6 released by IL-1β stimulation.

TABLE 2 ELISA Results Antibody IC₅₀ (nM) AB-control 0.017 AB1(invention) 0.15 AB5 (invention) 0.014

These results demonstrate the in vitro potency of the antibodies of theinvention to inhibit IL-1β. Furthermore, inhibition of IL-1-stimulatedcytokine release in MRC 5 has been shown to correlate with the agent'sability to inhibit IL-1 mediated activity in vivo. Thus, these resultsindicate that the antibodies of the invention will have IL-1 inhibitoryefficacy in vivo.

Example 3

This example illustrates the in vivo inhibition of IL-1β usingantibodies of the invention.

To confirm the in vivo efficacy of AB5, its ability to block thebiological activity of human IL-1β was tested in mice. Details of theassay are described in Economides et al., Nature Med., 9: 47-52 (2003).Briefly, male C57/B16 mice (Jackson Laboratory Bar Harbor, Me.) wereinjected intraperitoneally with titrated doses of AB5 (Example 1),AB-control (Example 1), or control IgG (Jackson ImmunoResearchLaboratories, West Grove, Pa.). Twenty-four hours after antibodyinjection, mice were injected subcutaneously with recombinant human IL-1(rhIL-1β) (from PeproTech Inc., Rocky Hill, N.J.) at a dose of 1 μg/kg.Two hours post-rhIL-1β injection (peak IL-6 response time), mice weresacrificed, and blood was collected and processed for serum. Serum IL-6levels were assayed by ELISA (BD Pharmingen, Franklin Lakes, N.J.)according to the manufacturer's protocol. Percent inhibition wascalculated from the ratio of IL-6 detected in experimental animal serumto IL-6 detected in control (multiplied by 100).

The results are set forth in FIG. 6. The ability to inhibit the in vivoactivity of IL-1β is assessed as a function of IL-1β stimulated IL-6levels in serum. As illustrated by FIG. 6, the AB5 antibody was aseffective, if not more effective, than AB-control for inhibiting the invivo activity of human IL-1β. 3 μg of AB5 was as effective as a 10 μg ofAB-control in this assay.

Thus, the results indicate that the tested antibodies are useful for theinhibition of IL-1β activity in vivo. These results also show that asingle injection of AB5 can block the systemic action to IL-1βstimulation over a prolonged period of time.

Example 4

The following example illustrates the preparation of an antibody inaccordance with the invention.

A number of human engineered antibody sequences were generated usingHUMAN ENGINEERING™ technology as described in Studnicka et al., ProteinEngineering, 7: 805-814 (1994), and in U.S. Pat. Nos. 5,766,886,5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication WO93/11794. Generated human engineered antibody sequences include AB5.1,AB5.2, AB5.3 and AB5.4. As shown in FIGS. 3 and 4, each of thesesequences comprise two variable positions in the CDR-3H region indicatedby X₁ and X₂. Thus, in certain examples of each one of these humanengineered antibodies, X₁ and X₂ of the CDR3 correspond to alanine andarginine, valine and arginine, phenylalanine and arginine, lysine andlysine, or asparagine and arginine, respectively.

Example 5

Antibodies designated AB5 and AB7 (a human engineered antibody sequence)were assayed for IL-1β binding properties using a kinetic exclusionassay performed on a KINEXA™ device in a manner like that described inExample 1. Additional description about KINEXA™ devices and operationfor antibody characterization is available from the manufacturer and canbe found in the published literature, for example U.S. Pat. No.6,664,114 (Sapidyne, Inc.); and Darling et al., “Kinetic Exclusion AssayTechnology: Characterization of Molecular Interactions.” ASSAY and DrugDevelopment Technologies, 2004, 2, 647-657. The KINEXA™ device performsa kinetic exclusion assay, and fits the data to various theoreticalcurves and thus determines K_(D) as well as other properties, such as95% confidence intervals for K_(D). The KINEXA™ device is generally moresensitive than other devices (e.g., a BiaCore device) for analysis ofaffinity characteristics such as dissociation constants and off-rates.

The amino acid sequences of the heavy and light chain variable regionsof AB5 and AB7 are provided in FIGS. 3 and 4A, respectively. IL-1βbinding assay results are summarized in Table 3. As in Example 1, K_(D)was calculated as the ratio of “off rate” to “on rate”, and a lowerK_(D) rate is indicative of higher antibody affinity.

TABLE 3 Antibody K_(D) (pM) AB5 0.24 AB7 0.30

The results of these experiments show that AB5 (consistent with resultsobserved in Example 1) and AB7 bind IL-1β with unexpectedly highaffinity, which is represented by the unexpectedly low values for theirdissociation constants.

FIGS. 7, 8, and 9 show the binding affinities of antibodies designatedAB1, AB5, and AB7, respectively, as determined from one representativeexperiment for each using KINEXA analysis. FIG. 7 reflects the resultsset forth in Table 1, while FIGS. 8 and 9 reflects the results set forthin Table 3.

In addition to the values set forth in Table 3, the KINEXA assay resultsalso indicate low and high 95% confidence intervals (K_(D)-low andK_(D)-high). For AB5, K_(D)-low was 0.07 pM, and K_(D)-high was 0.72 pM.For AB7, K_(D)-low was 0.11 pM, and K_(D)-high was 0.74 pM.

Similar K_(D)-low and K_(D)-high values were found in the assay setforth in Example 1. For AB-control, K_(D)-low was 1.62 pM, andK_(D)-high was 5.23 pM. For AB1, K_(D)-low was 13.38 pM, and K_(D)-highwas 24.84 pM. For AB5, K_(D)-low was 0.11 pM, and K_(D)-high was 0.56pM.

The KINEXA assay results indicate that AB5 and AB7 had anunexpectedly-lower dissociation constant than AB-control.

Example 6

This example illustrates the in vitro inhibition of IL-1β stimulatedrelease of IL-6. IC₅₀ for inhibiting IL-1β stimulated release of IL-6from human fibroblasts was assayed for several antibodies of the presentinvention as follows.

The IL-1β inhibitory potency of AB5 and AB7 was evaluated in a mannerlike that described in Example 2, using a bioassay that measures theIL-1β stimulated release of IL-6 from human fibroblasts. FIGS. 10-12show binding curves for individual assays on various antibodies. FIG. 10shows the inhibition of IL-6 release from human fibroblasts byantibodies designated AB1, AB2 and AB3, and the results of these threeindividual assays indicated that AB1 had an IC₅₀ of 0.029 nM (29 pM),AB2 had an IC₅₀ of 0.076 nM (76 pM), and AB3 had an IC₅₀ of 0.214 nM(214 pM). FIG. 11 shows the inhibition of IL-6 release from humanfibroblasts by antibodies designated AB1 and AB7 in an additional assay.FIG. 12 shows the inhibition of IL-6 release from human fibroblasts byantibodies AB5 and AB7, as well as the commercially available Kineret®.The results indicated that AB5 and AB7 had substantially better potencywith respect to inhibiting IL-1β than Kineret®, based on IC₅₀determinations in the assays. Kineret® is a man-made protein that issimilar to a naturally occurring protein called interleukin-1 receptorantagonist (IL-1ra) found in the body. FIGS. 10-12 show individual assayresults for the potency of the antibodies or Kineret® in terms ofpercent inhibition of IL-6 without the antibody, and Table 4 shows theIC₅₀ calculated from those individual assays. IC₅₀ is the concentrationof antibody or Kineret® required to inhibit 50% of IL-6 released byIL-1β stimulation.

TABLE 4 Antibody IC₅₀ (Nm) AB5 0.0049 (4.9 pM) AB7 0.0044 (4.4 pM)Kineret 0.0454 (45.4 pM)

In addition to the individual assay results reported in Tables 2 and 4and shown in FIGS. 6, 10, 11 and 12, other individual assays wereconducted for each of AB1, AB7 and AB-control. A mean IC₅₀ can becalculated from individual assay results. A mean IC₅₀ for AB1 of 66.7 pMwas calculated from individual assay results of 35 pM, 30 pM, 150 pM(this value is also shown in Table 2), and 52 pM. A mean IC₅₀ for AB7 of5.6 pM was calculated from individual assay results of 7.3 pM, 4.2 pM,4.5 pM, 4.4 pM (this value is also shown in Table 4), 6.0 pM, 5.0 pM,and 7.8 pM. A mean IC₅₀ for AB-control of 8.9 pM was calculated fromindividual assay results of 5.0 pM, 17.0 pM (this value is also shown inTable 2), and 4.9 pM.

These results demonstrate the in vitro potency of the AB1, AB5 and AB7to inhibit IL-β. Furthermore, inhibition of IL-1β-stimulated cytokinerelease in human fibroblasts has been shown to correlate with theinhibiting agent's ability to inhibit IL-1 mediated activity in vivo.Thus, these results indicate that the antibodies of the invention willhave IL-1β inhibitory efficacy in vivo.

Example 7

This example illustrates the in vivo inhibition of IL-1β using IL-1βbinding antibodies.

The in vivo efficacy of AB5, AB1 and AB7 and their ability to block thebiological activity of human IL-1β were tested in mice in a manner likethat described in Example 3. Results from testing AB5 and AB1 are setforth in FIG. 13, and results from testing AB5 and AB7 are set forth inFIG. 14. The ability to inhibit the in vivo activity of IL-1β wasassessed as a function of IL-1β stimulated IL-6 levels in serum. Asillustrated by FIGS. 13 and 14, the AB1, AB5 and AB7 antibodies wereeffective for inhibiting the in vivo activity of human IL-1.

These results indicate that the tested antibodies are useful for theinhibition of IL-1β activity in vivo.

Example 8

This Example illustrates that at least some IL-1β binding antibodiesaccording to the present invention are cross-reactive with IL-1β fromsome mammals other than humans, and are not cross-reactive with IL-1βfrom other non-human mammals. Antibody designated AB7 (an antibody thatbinds to human IL-1β with high affinity) was assayed for binding toIL-1β from non-human mammals, namely rhesus macaque, cynomolgus monkey,dog, guinea pig, and rabbit.

Fresh heparanized whole blood from rhesus macaque, cynomolgus monkey,dog, guinea pig and rabbit was obtained from Charles River Labs. Thewhole blood was separated by Ficoll density gradient centrifugation andperipheral blood mononuclear cells (PBMC's) were isolated. For eachspecies' PBMC, 2.5×10⁵ cells/ml were incubated in peripheral RPMI mediawith and without 50 ng/ml Lipopolysaccharide LPS (E. Coli 055:B5), andsupernatants were collected at 24 hours post-stimulation. LPS isintended to stimulate the production of IL-1β by the PBMC's. 2 ml ofeach supernatant was incubated for 3 hours with 2 μg of AB7 followed byaddition of 50 μl protein A-Sepharose bead slurry to immunoprecipitatethe AB7/IL-1β complex. Human IL-1β (Peprotech) was spiked into RPMI andrun as immunoprecipliation/Western blot controls. After centrifugationand washing of the Protein A-Sepharose beads, all samples were loadedonto a SDS-PAGE gel and run at 120V for 1 hour. Following transfer toImmobilon-P membrane at 22V overnight and blocking with 5% nonfat milk,AB7 was incubated at 2 μg/ml with the membrane for 2 hours. A secondarygoat anti-human IgG antibody conjugated with horseradish peroxidase(HRP) was added following wash steps and detection was with one steptetramethyl benzidine (TMB) solution.

FIGS. 15 and 16 show the Western blots obtained from this procedure. Onthe left side of the blot shown in FIG. 15 (lanes 1-3) are the controlsin which varying amounts (5 ng, 10 ng, and 20 ng) of human IL-1β wereadded to the RPMI media. Near the bottom of the blot, bands can be seenin each of the lanes at a region corresponding to a molecular weight ofapproximately 17 kDa. These bands are indicative of the binding of AB7to human IL-1β. The middle lane (lane 4) is the RPMI media. On the rightside of the blot shown in FIG. 15 (lanes 5-8), the results for thesamples from cynomolgus monkey and rhesus macaque are shown. Lanes 5 and6 are the cynomolgus monkey samples without LPS and with 50 ng LPS addedto the RPMI media, respectively. Lanes 7 and 8 are the rhesus macaquesamples without LPS and with 50 ng LPS added to the RPMI media,respectively. Near the bottom of the Western blot, bands can be seen inLanes 6 and 8 (the samples to which LPS was added) at a regioncorresponding to a molecular weight of approximately 17 kDa. These bandsin Lanes 6 and 8 are indicative of cross-reactivity of AB7 with primateIL-1β, namely IL-1β from cynomolgus monkey and rhesus macaque.

FIG. 16 shows Western blots for controls and samples from PBMC's of dog,guinea pigs, and rabbits. On the left side of the blots shown in FIG. 16(lanes 1-4) are the controls in which varying amounts (5 ng, 10 ng, 50ng, and 200 ng) of human IL-1β were added to the RPMI media. Near thebottom of the blot, bands can be seen in each of the lanes at a regioncorresponding to a molecular weight of approximately 17 kDa. These bandsare indicative of the binding of AB7 to human IL-1β. Lane 5 in FIG. 15is the RPMI media. Lanes 6-8 are the results for the samples from dogPBMC's, with no LPS, 50 ng LPS and 200 ng LPS, respectively. Lanes 9 and10 are the results for the samples from guinea pig PBMC's, with no LPSand 50 ng LPS, respectively. Lanes 11 and 12 are the results for thesamples from rabbit PBMC's, with no LPS and 50 ng, respectively. Nearthe bottom of the Western blot, bands can be seen in Lanes 7, 8 and 12(the dog and rabbit samples to which LPS was added) at a regioncorresponding to a molecular weight of approximately 17 kDa. These bandsin Lanes 7, 8 and 12 are indicative of cross-reactivity of AB7 with dogIL-1β and rabbit IL-1β. The absence of a visible band in Lane 10 (guineapig PBMC with 50 ng LPS added) indicates that AB7 was not cross-reactivewith guinea pig IL-1β.

These results indicate that AB7 is cross-reactive with IL-1β fromseveral non-human mammals, namely rhesus macaque, cynomolgus monkey,dog, and rabbit, but is not cross-reactive with IL-1β from at least oneother non-human mammal, namely guinea pig.

Example 9

This Example further illustrates that at least some IL-1β bindingantibodies according to the present invention are cross-reactive withIL-1β from other non-human mammals. Antibody AB7 was assayed for bindingto IL-1β from non-human mammals, namely mouse and rat.

Recombinant human, mouse and rat IL-1β (Peprotech) were loaded inreducing and non-reducing condition onto a SDS-PAGE gel and run at 120Vfor 1 hr. Following transfer to Immobilon-P membrane at 22V overnightand blocking with 5% nonfat milk, AB7 was incubated at 2 μg/ml with themembrane for 2 hours. A secondary goat anti-human IgG HRP conjugatedantibody was added following wash steps and detection was with one stepTMB solution.

FIG. 17 shows the Western blot obtained by the foregoing procedures.Lanes 1 and 2 are for non-reduced and reduced human IL-1β, respectively.Lanes 3 and 4 are for non-reduced and reduced mouse IL-1β, respectively.Lanes 5 and 6 are for non-reduced and reduced rat IL-1β, respectively.At the bottom of the blot, bands can be seen in each of the lanes at aregion corresponding to a molecular weight of approximately 17 kDa.These bands are indicative of the presence IL-1β, which in turn isindicative of the binding of AB7 to human IL-1β mouse IL-1β and ratIL-1β. These results indicate that AB7 is cross-reactive with rodentIL-1β.

Example 10

This Example further illustrates that at least some IL-1β bindingantibodies according to the present invention are inhibitors of IL-1βfrom humans and at least some non-human mammals. Antibody AB7 wasassayed for inhibiting the proliferation of D 0 cells stimulated byhuman, rhesus macaque, mouse and rat IL-1β.

D10.G4.1 (D10) cells are murine T helper cells with specificity for theconalbumin antigen from egg white. This cell line was derived from theAKR/J mouse (H-2^(k) MHC haplotype) and requires IL-1β and antigenreceptor activation for growth, proliferation, and survival. The D10cell line is highly sensitive to IL-1 and can respond to IL-1 fromseveral species (including human, monkey, mouse, and rat) which allowsfor testing the cross-reactive neutralizing potential of an IL-1βbinding antibody or fragment, such as AB7. D10 proliferation is notaffected by LPS or by macrophage-derived cytokines such as IL-6 andTNF-a. As a result, D10 assays can be used to assess the specific IL-1activity from endogenous sources (i.e., LPS-activated macrophages).

D10 cells were activated with Concanavalin A (Con A) and a constantlevel of recombinant or native source of IL-1 in the presence or absenceof several concentrations of AB7. Cells were plated at 2×10⁴/well andstimulated with 2.5 μg/ml Con A and different concentrations of IL-1β.Cells were cultured for 72 hours and proliferation was measured byadding the redox viability dye Alamar Blue during the last 8-14 hours ofculture and assessing the O.D.₅₇₀₋₆₀₀.

To test the potency and species cross-reactivity of AB7, the D10bioassay was performed using the following concentrations of recombinantor native IL-1β: 10 pg/ml recombinant human IL-1β; 10 pg/ml recombinantrhesus IL-1β; 10 pg/ml mouse IL-1β; and 100 pg/ml rat IL-1β. For the D10assay employing endogenous human IL-1, a 1:360 dilution of supernatantfrom LPS-activated human PBMC's was used. Different concentrations ofAB7 were tested with each IL-1β. IC₅₀ measurements were determined usingGraphpad Prism. Mean, standard deviation (SD), and standard error (SEM)for IC₅₀ were calculated using Microsoft Excel.

Results from the D10 assay are summarized in Table 5, which includes themean IC₅₀ and the SEM (based on 4 experiments for recombinant humanIL-1β and 3 experiments for the IL-1β from other sources). AB7 washighly potent in neutralizing recombinant human IL-1β and endogenouslyproduced (native) human IL-1β. AB7 was also highly potent inneutralizing recombinant rhesus macaque IL-1β. AB7 also neutralizedrecombinant mouse IL-1β with lower potency, having an IC₅₀ that was1000-fold higher compared to human. AB7 did not have significantactivity against rat IL-1β in this assay.

TABLE 5 ELISA Results IC₅₀ (pM) SEM (pM) recombinant human IL-1β 2.4±0.52 endogenously produced (native) human IL-1β 2.6 ±0.11 recombinantrhesus macaque IL-1β 2.7 ±0.73 recombinant mouse IL-1β 2618 ±60.9

These results indicate that AB7 is a highly potent neutralizing antibodyagainst human IL-1β with similar potency against recombinant and nativeforms of the cytokine. Activity against the non-human primate rhesusmacaque IL-1β was similar as that against human IL-1β. Thus, at leastsome antibodies and fragments of the present invention encompassantibodies and fragments having substantially the same potency againsthuman IL-1β and primate IL-1β and/or having substantially the samepotency against recombinant human IL-1β and endogenous human IL-1β.These results also indicate that AB7 also neutralizes mouse IL-1β.

Example 11

This Example illustrates the mapping of the IL-1β epitope to which atleast some antibodies of the present invention (for example, theantibody designated AB7) bind.

A PepSpot™ peptide array (JPT Peptide Technologies, Berlin, Germany) wasused to identify the IL-1β key amino-acid residues (epitope) involved inthe binding of AB7. A scan of twelve amino-acid peptides, spanning theentire IL-1β amino-acid sequence, each peptide overlapping by 11 aminoacid to the previous one, were synthesized directly on a membrane. Themembrane carrying the peptides was probed with AB7 at a concentration of2 μg/ml, for 2 hr at room temperature. Binding of AB7 to membrane boundpeptides was detected using a secondary HRP-conjugated goat anti-humanantibody, followed by enhanced chemiluminescence (ECL). The peptidesspots corresponding to IL-1β residues 83-105 scored positive for bindingto AB7.

This mapping indicates that AB7 binds to an epitope within the sequencecorresponding to residues 83-105 of the mature IL-1β protein. Thesequence comprises the amino acids ESVDPKNYPKKKMEKRFVFNKIE, and AB7 isexemplary of antibodies that bind to an epitope within this sequence. Itis expected that the antibodies designated AB6, AB8, AB9, and others,such as antibodies having the heavy chain of SEQ ID NO:29 and the lightchain of SEQ ID NO:27, also bind to an epitope contained in thissequence.

Example 12

This example illustrates the in vitro inhibition of IL-1β using anantibody of the invention in an cell based assay IL-8.

Fresh, heparinized peripheral blood was collected from healthy donors.180 μl of whole blood was plated in a 96-well plate and incubated withvarious concentrations of the antibody AB7 and 100 pM rhIL-10. ForKineret®-treated samples, Kineret® and rhIL-1β were combined 1:1 priorto mixing with blood. Samples were incubated for 6 hours at 37° C. with5% CO₂. Whole blood cells were then lysed with 50 μl 2.5% Triton X-100.The concentration of interleukin-8 (IL-8) in cleared lysates was assayedby ELISA (Quantikine human IL-8 ELISA kit, R&D Systems) according tomanufacturer's instructions. IL-8 concentrations in AB7 and Kineret®treated samples were compared to a control sample treated with anti-KLHcontrol. The results are depicted in FIG. 18 and summarized in Table 6.IC₅₀ is the concentration of antibody required to inhibit 50% of IL-8released by IL-1β stimulation.

TABLE 6 IC₅₀ (pM) AB7  1.9 pM Kineret ® 53.4 pM

These results demonstrate the in vitro potency of the AB7, as measuredby inhibition of IL-1β stimulated release of IL-8. These results showinggreater potency compared with Kineret® indicate that the antibodies ofthe invention will have IL-1β inhibitory efficacy in vivo.

Example 13

This example illustrates that the antibodies of the invention havesurprisingly high affinity in comparison to an antibody having a similarsequence.

AB5 was compared to AB-control in terms of sequence and bindingaffinity. AB5 comprises the heavy chain variable region set forth in SEQID NO:8 and the light chain variable region set forth in SEQ ID NO:9.AB-control is believed to comprise the heavy chain variable region setforth in SEQ ID NO:38 and the light chain variable region set forth inSEQ ID NO:39. Those sequence set forth in U.S. Patent ApplicationPublication No. 2003/0026806, at FIGS. 6A and 6B. AB5 and AB-controlhave the same complementarity determining regions in their heavy andlight chain variable regions. Their heavy chains differ by three aminoacid residues in framework region 3, located at positions 68, 74 and 86in SEQ ID NOS: 8 and 38. Their respective light chains differ by oneamino acid residue in framework region 3, located at position 72 in SEQID NOS: 9 and 39. Despite the similarities in the sequences of theirheavy and light chain variable regions, including the same CDRs, AB5 andAB-control differ significantly and unexpectedly in their bindingaffinity. As discussed in Examples 1 and 5 above, AB5 was found to havea dissociation constant of less than 0.3 pM (with a K_(D)-low of 0.11pM, and a K_(D)-high of 0.56 pM), and AB-control was found to have adissociation constant of 3 pM (with a K_(D)-low of 1.62 pM, and aK_(D)-high of 5.23 pM). Given the similarities in amino acid sequence,it is surprising that AB5 has higher affinity by an order of magnitude.

AB7 was generated using HUMAN ENGINEERING technology, as described inExample 4. The light and heavy chain variable regions of AB7 include lowand moderate risk positions in the sequences of light and heavy chainvariable regions AB5. AB7 comprises the heavy chain variable region setforth in SEQ ID NO:15 and the light chain variable region set forth inSEQ ID NO:11.

AB7 was compared to AB-control and AB5 in terms of sequence and bindingaffinity. AB7 and AB-control have the same complementarity determiningregions in their heavy and light chain variable regions. Their heavychains differ at two of the three positions in framework region 3(positions 74 and 86 in SEQ ID NOS: 15 and 38) where AB5 differed fromAB-control; however, at position 68 in SEQ ID NO: 15, AB7 has the sameamino acid as AB-control. In the light chain of AB7, position 72 in SEQID NO: 11 differs from both AB-control and AB5. AB7 includes severalother differences in the light and heavy chain variable regions whencompared to AB-control and AB5 by virtue of the HUMAN ENGINEERING™process. Despite the inclusion of changes at moderate risk positions,and particularly in view of the changes in AB7 compared to AB5 atposition 68 in the heavy chain variable region and at position 72 in thelight chain variable region, AB7 and AB5 have similar dissociationconstants, and AB7 differs significantly and unexpectedly fromAB-control with respect to binding affinity. As discuss in Example 5above, AB7 was found to have a dissociation constant of 0.3 pM (with aK_(D)-low of 0.11 pM, and a K_(D)-high of 0.74 pM). AB5 was found tohave a dissociation constant of 0.24 pM (with a K_(D)-low of 0.07 pM,and a K_(D)-high of 0.72 pM). Given the changes made in moderate riskpositions, and the overall similarities in amino acid sequence,particularly in the CDRs, it is surprising that AB7 has similar affinityto AB5 and higher affinity than AB-control by an order of magnitude.

Example 14

This example shows that at least one antibody of the present inventionbinds to an IL-1β epitope such that the bound antibody does notsubstantially prevent the antibody-bound IL-1β from binding to IL-1receptor type I. This Example employs a Biacore® kinetic analysisinstrument to examine whether IL-1β bound to one of the presentantibodies (AB7) can still bind to IL-1 receptor type I.

For this Example, AB7 was immobilized on the surface of a CM-5 sensorchip in a Biacore instrument as follows. Using HBS-EP (Biacore®, Inc.)as running buffer, the temperature was set to 25° C., the flow rate wasset to 10 μL/min, and the flow path was directed to flow cell 2 only.135 μL of each of NHS and ECD solutions (Biacore®, Inc.) were mixed, and70 μL of the NHS/ECD solution was immediately injected in the flow path.Then 91 μL of an AB7 solution (20 μg/mL in 10 mM sodium acetate buffer(Biacore®, Inc.)) was injected, followed by 70 μL of 1M Ethanolamine(Biacore®, Inc.). Approximately 5650 RU of AB7 was thus immobilized. Toprepare a reference surface, the flow path was changed to flow cell 1only. 135 μL of each of NHS and ECD solutions (Biacore®, Inc.) weremixed, and 70 μL of the NHS/ECD solution was immediately injected in theflow path, followed by 70 μL 1M Ethanolamine (Biacore®, Inc.).

The Biacore® instrument was then ready for the analysis of whether IL-1βbound to AB7 would still bind to IL-1 receptor type I. For thisanalysis, soluble IL-1β receptor type I (IL-1 sRI) was used, and thebinding of IL-1 sRI to a complex of AB7/IL-1β was tested as follows.IL-1 sRI (RnDSystems cat#269-1R-100/CF) and IL-1β (Peprotech,cat#200-01B) were separately diluted to 10 ug/mL in HBS-EP. The flow atewas set to 10 μL/min. The flow path for the Biacore® instrument was setto flow cells 1 and 2, and reference subtraction of flowcell 2 fromflowcell 1 was used to determine a response differential. FIG. 19 showsthe measured response differential from the Biacore® instrument over thecourse of the analysis. FIG. 20 provides an illustration of the stepsused in the analysis, indicating the separate additions to the flow cellof (A) IL-1 sRI, (B) IL-1β, and (C) IL-1 sRI, in that order.

At 200 seconds, 20 μL of IL-1 sRI was injected to verify absence ofbinding directly to immobilized AB7 (injection A in FIGS. 19 and 20). Asshown in FIG. 19, IL-1 sRI did not increase the response units, whichindicates that IL-1 sRI did not bind directly to the immobilized AB7.

At 600 seconds, approximately 1000 RU of IL-1b was bound by AB7, forminga AB7/IL-10 complex on the chip surface (injection B in FIGS. 19 and20). The increase in response units indicates that the IL-1β bound tothe immobilized AB7. 20 μL of IL-1 sRI was next injected at 1200 secondsto test binding of IL-1 sRI to the complex of AB7 and IL-1β.Approximately 1500 RU of IL-1 sRI bound to the AB7/IL-1β complex(injection C in FIGS. 19 and 20). This increase in response unitsindicates that IL-1 sRI bound to the IL-1β/AB7 complex.

This Example indicates that IL-1 sRI binds to IL-10 but does not bind toAB7, and that AB7 binds to an IL-1β epitope such that the bound AB7 doesnot substantially prevent the IL-1β from binding to IL-1 sRI.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Wherever an open-ended term isused to describe a feature or element of the invention, it isspecifically contemplated that a closed-ended term can be used in placeof the open-ended term without departing from the spirit and scope ofthe invention. Recitation of ranges of values herein are merely intendedto serve as a shorthand method of referring individually to eachseparate value falling within the range, unless otherwise indicatedherein, and each separate value is incorporated into the specificationas if it were individually recited herein. All methods described hereincan be performed in any suitable order unless otherwise indicated hereinor otherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseworking in the art upon reading the foregoing description. The inventorsexpect skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. An isolated IL-1β binding antibody or IL-1β binding fragment thereofcomprising a heavy chain variable region of SEQ ID NO: 8 and a lightchain variable region of SEQ ID NO:
 9. 2. The antibody or antibodyfragment of claim 1, wherein the antibody or antibody fragment comprisesa lambda light chain.
 3. The antibody or antibody fragment of claim 1,wherein the antibody or antibody fragment comprises an IgG2 region. 4.The antibody or antibody fragment of claim 1, wherein the antibody orantibody fragment is an Fab, an F(ab′)2, an Fv, or a single-chainantibody fragment.
 5. An isolated IL-1β binding antibody or IL-1βbinding fragment thereof, wherein the antibody or fragment binds tohuman IL-1β with a dissociation constant less than 1 pM, and theantibody or fragment competes with the binding of an antibody comprisingthe light chain variable region of SEQ ID NO: 9 and the heavy chainvariable region of SEQ ID NO:
 8. 6. A composition comprising (a) theantibody or antibody fragment of claim 1 or 5, and (b) a suitablecarrier.
 7. The composition of claim 6, wherein the carrier is apharmaceutically acceptable carrier.
 8. The composition of claim 7,wherein the composition is in a form suitable for intraarticular,subcutaneous, intravenous, intraperitoneal, intracerebral,intraparenchymal, intracerebroventricular, intramuscular, intraocular,intraarterial, intralesional, oral or inhalation administration.
 9. Thecomposition of claim 7, wherein the composition comprises alyoprotectant, a surfactant, a filler, a binder, and/or bulking agent.10. The composition of claim 7, wherein the composition is acontrolled-release or sustained-release pharmaceutical composition.